Note: Descriptions are shown in the official language in which they were submitted.
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SUSTAINED RELEASE L-ARGININE
FORMULATIONS AND METHODS OF MANUFACTURE AND USE
Related Applications
This application claims priority to U.S. Provisional Patent Application Serial
No.
60/507,312, entitled "Methods and Compositions for the Treatment of
Cerebrovascular
and Cardiovascular Diseases and Disorders" filed September 29, 2003, U.S.
Provisional
Patent Application Serial No. 60/512,035, entitled "Sustained Release L-
Arginine
l0 Formulations and Methods of Manufacture and Use" filed October 17, 2003,
and
PCT/LJS2003/033931, entitled "Sustained Release L-Arginine Formulations and
Methods of Manufacture and Use" filed October 24, 2003. This application is
related to
U.S. Provisional Patent Application Serial No. 60/421,258, entitled "Methods
and
Compositions for the Treatment of Cerebrovascular and Cardiovascular Diseases
and
15 Disorders" filed October 24, 2002. The entire contents of each of the
aforementioned
applications are hereby expressly incorporated herein by reference in their
entireties.
Background of the Invention
A family of enzymes called nitric oxide syntheses (NOS) synthesize nitric
oxide
20 (NO), an important biological second messenger, from L-arginine. There are
several
distinct isoforms of NOS including constitutive NOS (cNOS) and inducible NOS
(iNOS). There are two different kinds of cNOS: endothelial NOS (eNOS) and
neuronal
NOS (nNOS). eNOS is involved in the regulation of smooth muscle relaxation,
blood
pressure lowering, and inhibition of platelet aggregation. eNOS resides in
endothelial
25 cells and releases NO over short periods in response to receptor-mediated
increases in
cellular Ca2+. Michel et al., "Nitric oxide syntheses: which, where, how, and
why?," J.
Clin. Invest 100: 2146-2152 (1997). nNOS is important for long-term
potentiation, and
is responsible for the Ca2+ dependent release from neurons. iNOS acts in host
defense, is
generated by activated macrophage cells during an immune response, is induced
in
3o vascular smooth muscle cells (e.g., by various cytokines, microbial
products, and/or
bacterial endotoxins), and once expressed, synthesizes NO for long periods of
time.
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Formation of nitric oxide by cNOS in endothelial cells is thought to play an
important role in normal blood pressure regulation, prevention of endothelial
dysfunction such as hyp.erlipidemia, arteriosclerosis, thrombosis, and
restenosis.
Functionally, cNOS, which is the predominant synthase present in brain and
endothelia,
is active under basal conditions and can be further stimulated by increases in
intracellular calcium that occur in response to receptor-mediated agonists or
calcium
ionophores. cNOS appears to be the "physiological" form of the enzyme and
plays a
role in a diverse group of biological processes. Ih vitro studies suggest that
the activity
of NOS can be regulated in a negative feedback manner by nitric oxide itself.
In
to cardiocerebrorenovasculax circulation, the primary target for
constitutively produced NO
is believed to be soluble guanylate cyclase located in vascular smooth muscle,
the
myocardium (myocytes) and coronary vascular smooth muscle.
In contrast to cNOS, the inducible, calcium-independent isoform, iNOS was
initially only described in macrophages. It is now known that induction of
nitric oxide
synthase can occur in response to appropriate stimuli in many other cell
types. This
induction occurs both in cells that normally do not express a constitutive
form of nitric
oxide synthase, such as vascular smooth muscle cells, as well as in cells such
as those of
the myocardium that express considerable levels of the constitutive isoform.
iNOS exhibits negligible activity under basal conditions, but in response to
2o factors such as lipopolysaccharide and certain cytokines, expression occurs
over a period
of hours. The induced form of the enzyme produces much greater amounts of NO
than
the constitutive form, and induced NOS appears to be the "pathophysiological"
form of
the enzyme because high concentrations of iNOS produced NO can be toxic to
cells.
Induction of iNOS can be inhibited by glucocorticoids and some cytokines.
Relatively
little is known about post-transcriptional regulation of iNOS. Cytotoxic
effects due to
NO are probably largely independent of guanylate cyclase and cyclic GMP
formation.
Most of the research in this area has focused on the stimulation of iNOS
inhibitors using
various derivatives of L-arginine.
NO is a relatively stable free radical synthesized from molecular oxygen and
the
3o guanidino nitrogen of L-arginine in a reaction catalyzed by NOS. This
enzyme is found
in many tissues and cell types including neurons, macrophages, hepatocytes,
smooth
muscle cells, endothelial cells of the blood vessels, and epithelial cells of
the kidney.
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NO acts near its point of release, entering the target cell and activating the
cytosolic
enzyme guanylate cyclase, which catalyzes the formation of the second
messenger cyclic
GMP (cGMP). Within seconds of the formation of NO, it undergoes oxidation to
nitrite
or nitrate. David L. Nelson, Michael M. Cox, Lehninger Principles of
Biochemistry, p.
892, 3rd ed. Worth Publishers, 2000.
In response to a variety of vasoactive agents and even physical stimuli, the
endothelial cells release a short-lived vasodilator called endothelium derived
relaxing
factor (EDRF) (also referred to as endothelium derived nitric oxide (EDNO)).
Products
of inflammation and platelet aggregation such as serotonin, histamine,
bradykinin,
to purines, and thrombin exert all or part of their action by stimulating the
release of NO.
Endothelial cell-dependent mechanisms of relaxation are important in a variety
of
vascular beds, including the coronary circulation. Hobbs et al., Annu. Rev.
Pharmacol.
Toxicol. 39: 191-220 (1999). NO diffuses readily to the underlying smooth
muscle and
induces relaxation of vascular smooth muscle by activating guanylate cyclase,
which
15 increases cGMP concentrations.
NO is responsible for the endothelium dependent relaxation and activation of
soluble guanylate cyclase, neurotransmission in the central and peripheral
nervous
systems, and activated macrophage cytotoxicity. In the vasculature, EDNO has
several
actions among which are the inhibition of platelet aggregation, adhesion of
inflammatory
20 cells, and the proliferation of smooth muscle cells. In particular, EDNO is
an important
regulator of vascular tone. Also, flow dependent dilation, a commonly used
index of
endothelial function, is largely mediated by NO.
The mechanism for the regulation of vascular tone by NO is initiated by
stimuli,
such as acetylcholine, bradykinin, shear stress, etc., on the endothelial
cells lining the
25 vasculature. NO is produced from L-arginine through the catalytic activity
of eNOS
contained in these endothelial cells. The NO produced leaves the endothelial
cells and
stimulates the guanylate cyclase activity in the adjoining smooth muscle
cells.
Activation of guanylate cyclase increases the level of cGMP and causes the
smooth cells
to relax, thus dilating the vessel and increasing the blood flow. Moncada et
al., New
3o Eng. J. Med. 329: 2002-2012 (1993); Vallance et al., J. Royl. Coll.
Physician London
28: 209-219 (1994).
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Summary of the Invention
The present invention provides methods for the treatment and prevention of
vascular diseases and disorders including, but not limited to, cardiovascular,
cerebrovascular and peripheral vascular diseases and disorders. The present
invention is
based, at least in part, on :the discovery that the coadministration of an HMG-
CoA
reductase inhibitor and a sustained release formulation of L-arginine has a
synergistic
effect in the treatment and prevention of vascular diseases and disorders,
and, in
particular, in lowering cholesterol and triglycerides. Moreover, the invention
provides a
sustained release formulation of L-arginine and methods of manufacture that
render a
l0 composition with an optimal release profile. Furthermore, the formulation
and methods
of manufacture render a composition that is conveniently compressible, but not
excessively friable.
In one aspect, the invention provides for a sustained release L-arginine
composition including about 50% to about 90% by weight of L-arginine or a
pharmaceutically acceptable salt thereof; about 0.5% to about 5% by weight of
polyvinylpyrrolidone; and about 5% to about 40% by weight of hydroxypropyl
rnethylcellulose. In a particular embodiment, the composition includes about
70% by
weight of L-arginine monohydrochloride, where the L-arginine is L-arginine
monohydrochloride; about 2% to about 3% by weight of polyvinylpyrrolidone; and
about
27% to about 2~% by weight of hydroxypropyl methylcellulose.
In another aspect, the invention provides for a sustained release L-axginine
composition including about 35% to about 90% by weight of L-arginine or a
pharmaceutically acceptable salt thereof; about 0.5% to about 5% by weight of
polyvinylpyrrolidone; about 5% to about 40%~by weight of hydroxypropyl
methylcellulose; about 2% to about 20% by weight of microcrystalline
cellulose; and
less than about 1% by weight of silicon dioxide: In one particular embodiment,
the
composition includes about 51 % by weight of L-arginine monohydrochloride,
where the
L-arginine is L-arginine monohydrochloride; about 3% to about 4% by weight of
polyvinylpyrrolidone; about 35% by weight of,hydroxypropyl methylcellulose;
about
10% to about 11 % by weight of microcrystalline cellulose; and less than about
1 % by
weight of colloidal ilicon dioxide, where the silicon dioxide is colloidal
silicon dioxide.
In yet another embodiment, the composition includes about: 56% by weight of L-
arginine
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~monohydrochloride, where the L-arginine is L=arginine monohydrochloride;
about 3% to
;about 4% by weight of polyvinylpyrrolidone; about 31 % to about 32% by weight
of
hydroxypropyl methylcellulose; about 9% to about 10% by weight of
microcrystalline
cellulose; and less than about 1% by weight of colloidal silicon dioxide,
where the
silicon dioxide is colloidal silicon dioxide.
In another aspect, the invention provides for a sustained release L-arginine
composition including about 50% to about 90% by weight of L-arginine or a
pharmaceutically acceptable salt thereof; about 0.5% to about 10% by weight of
polyvinylpyrrolidone; about 5% to about 40% by weight of hydroxypropyl
methylcellulose; and less than about 1 % by weight of silicon dioxide. In a
particular
embodiment, the composition includes about 69% by weight of L-arginine
mono'hydrochloride, where the L-arginine is L-arginine monohydrochloride;
about 6% to
about 7% by weight of polyvinylpyrrolidone; about 24% to about 25% by weight
of
hydroxypropyl methylcellulose; and less than about 1 % by weight of colloidal
silicon
dioxide, where the silicon dioxide is colloidal silicon dioxide.
In yet another aspect, the invention provides for a sustained release L-
arginine
composition, including about 35% to about 70% by weight of L-arginine or a
pharmaceutically acceptable salt thereof; about 0.5% to about 10% by weight of
polyvinylpyrrolidone; about 40% to about 60% by weight of hydroxypropyl
2o methylcellulose; and less than about 1 % by weight of silicon dioxide. In a
particular
embodiment, the composition includes about 50% by weight of L-arginine
monohydrochloride, where the L-arginine is L-arginine monohydrochloride; about
4% to
about 5% by weight of polyvinylpyrrolidone; about 45% by weight of
hydroxypropyl
,methylcellulose; and less than about 1 % by weight of colloidal silicon
dioxide, where the
silicon dioxide is colloidal silicon dioxide.
In another aspect, the invention provides for a method for lowering
cholesterol in
a subject including administering to a subject any of the sustained release
formulations
of the invention. In various embodiments, the method rnay lower total
cholesterol, low
density lipoprotein (LDL) cholesterol, and/or triglycerides, andlor increase
high density
lipoprotein (HDL) cholesterol in the subject. In another aspect, the invention
provides a
method for treating or preventing Alzheimer's disease, including administering
to a
subject any of the sustained release formulations of the invention. In yet
another aspect,
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the invention provides a method for treating or preventing intermittent
claudication,
including administering to a subject any of the sustained release formulations
of the
invention. In yet another aspect, the invention provides a method for lowering
C-
reactive protein, including administering to a subject any of the sustained
release
formulations of the invention.
In various other aspects, the present invention provides a method for treating
or
preventing a vascular disease or disorder, a method for treating or preventing
atherosclerosis, a method for increasing vasodilation, and/or a method for
increasing
nitric oxide production, including administering to a subject any of the
sustained release
1 o formulations of the invention.
In another aspect, the invention provides methods for lowering C-reactive
protein
in a subject including administering to a subject HMG-CoA reductase inhibitor
and a
sustained release formulation as described above. The method lowers C-reactive
protein
in a subject to a greater extent than merely administering HMG-CoA reductase
inhibitor
alone, or L-arginine alone.
In other aspects, the invention provides for a method for making a sustained
release composition of L-arginine by granulating the L-arginine, where L-
arginine is
about 70% by weight of the sustained release formulation, with a granulating
agent
including polyvinylpyrrolidone, where polyvinylpyrrolidone is between about 2%
and
2o about 3% by weight of the sustained release formulation; wet milling the
granules;
drying the granules; dry milling the granules; and blending the granules with
hydroxypropyl methylcellulose, where the hydroxypropyl methylcellulose is
about 27%
to about 2~% by weight of the sustained release formulation.
In another aspect, the invention provides for a method for making a sustained
release composition of L-axginine by granulating L-arginine, where L-arginine
is about
51 % by weight of the sustained release formulation, with a granulating agent
including
polyvinylpyrrolidone, where polyvinylpyrrolidone is between about 3% and about
4% by
weight of the sustained release formulation; wet milling the granules; drying
the
granules; dry milling the granules; and blending the granules with
hydroxypropyl
3o methylcellulose, wherein the hydroxypropyl methylcellulose is about 35% by
weight of
the sustained release formulation. The granules may be further blended with
microcrystalline cellulose and colloidal silicon dioxide, where the
microcrystalline
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cellulose is about 10% to about 11 % by weight of the sustained release
formulation, and
where the colloidal silicon dioxide is less than about 1 % by weight of the
sustained
release formulation.
In yet another aspect, the invention provides for a method for making a
sustained
release composition of L-arginine, comprising granulating L-arginine, where
the L-
argiiune is about 56% by weight of the sustained release formulation, with a
granulating
agent including polyvinylpyrrolidone, where the polyvinylpyrrolidone is
between about
3% and about 4% by weight of the sustained release formulation; wet milling
the
graamles; drying the granules; dry milling the granules; and blending the
granules with
hydroxypropyl methylcellulose, where the hydroxypropyl methylcellulose is
about 31%
to about 32% by weight of the sustained release formulation. In a particular
embodiment, the granules may be further blended with microcrystalline
cellulose and
colloidal silicon dioxide, where the microcrystalline cellulose is about 9% to
about 10%
by weight of the sustained release formulation, and where the colloidal
silicon dioxide is
less than about 1 % by weight of the sustained release formulation.
In yet another aspect, the invention provides for a'method for making a
sustained
release composition of L-arginine by granulating L-arginine, where the L-
arginine is
about 69% by weight of the sustained release formulation, with a granulating
agent
including polyvinylpyrrolidone, where the polyvinylpyrrolidone is between
about 6%
and about 7% by weight of the sustained release formulation; wet milling the
granules;
drying the granules; dry milling the graamles; and blending the granules with
hydroxypropyl methylcellulose, where the hydroxypropyl methylcellulose is
about 24%
to about 25% by weight of the sustained release formulation. In one
embodiment, the
granules may be further blended with colloidal silicon dioxide, where the
colloidal
silicon dioxide is less than about 1 % by weight of the sustained release
formulation.
In yet another aspect, the invention provides for a'method for making a
sustained
release composition of L-arginine by granulating L-arginine, where the L-
arginine
includes about 50% by weightof the sustained release formulation; with
granulating
agent including polyvinylpyrrolidone, where the polyvinylpyrrolidone is
between about
4% and about 5% by weight of the sustained release formulation; wet milling
the
granules; drying the granules; dry milling the granules; and blending the
granules with
hydroxypropyl~methylcellulose, where the hydroxypropyl methylcellulose is
about 45%
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by weight of the sustained :release formulation. In a particular embodiment,
the granules
may be further blended with colloidal silicon dioxide, where the colloidal
silicon dioxide
is less than about 1 % by weight of the sustained release formulation.
In particular embodiments of these methods of making the sustained release
5; formulation,'the blending step may include the steps of pre-blending,
blending and final
blending the granules. In other embodiments of these methods, the L-arginine
may be
dry mixed with the polyvinylpyrrolidone prior to the granulating step.
Other features and advantages of the invention will be apparent from the
following detailed description and claims.
10.
Brief Description of the Drawings
Figure 1 is a graph depicting the release pattern of a formulation comprising
L-
arginine and simvastatin.
Figure 2 is;photograph of NMR images of infarct size in a mouse brain treated
15 with L-arginine and simvastatin versus in an untreated mouse brain.
Figuf~e 3 is a bar graph depicting infarct volume in mice treated with L-
argiiune,
simvastatin and both L-arginine and simvastatin.
Figure 4 is a bar graph depicting total infarct volume in mice treated with L-
arginine and various levels of simvastatin.
20 Figure 5 is a flow chart depicting a method of manufacture of sustained
release
L-arginine tablets.
Figure 6 is a flow chart depicting a method of manufacture of sustained
release
L-arginine tablets.
Figuf~e 7 is a bar graph comparing the performance of sustained release L-
25 arginine formulations.
FiguYe 8 is a chart comparing the affect of administration of simvastatin with
and
without a sustained release L-arginine composition of the present invention on
endothelium-dependent vasodilation in humans.
Figure 9 is a chart summarizing the synergistic effect of administration of
30 simvastatin and a sustained release L-arginine composition of the invention
on
cholesterol levels in humans.
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Figuf°e 10 is a bar graph demonstrating the effect of simvastatin on
cultured
human aortic endothelial cells (HAEC) versus untreated cultured HAEC..
Detailed Description of the Invention
The present invention provides meth~ds for the treatment and prevention of
vascular diseases and disorders including, but not limited to, cardiovascular,
cerebrovascular and peripheral vascular diseases and disorders. The present
invention is
based, at least in part, on the discovery that the coadministration of an HMG-
CoA
reductase inhibitor and a sustained release formulation of L-arginine has a
surprising
to synergistic effect in the treatment and prevention of vascular diseases and
disorders
(including cerebrovascular, cardiovascular and peripheral vascular diseases or
disorders),
and, in particular,'in lowering cholesterol. Moreover, the sustained release L-
arginine
and, optionally, the HMG-CoA reductase inhibitor, may be used to increase
vasodilation,
increase NO production, and lower C-reactive protein. hi another embodiment,
the
formulations and methods described herein may be used to delay the onset of
the disease,
disorder and/or event in, for example, populations at risk for development of
vascular
diseases or disorders and/or an occurrence of an event. The HMG-CoA reductase
inhibitor and the sustained release formulation of L-arginine may be
administered to the
subject either sequentially or concurrently. The reductase inhibitor and the L-
arginine
may be contained within a single formulation.
Moreover, the invention provides a sustained release formulation of L-arginine
and methods of manufacture that render a composition with an optimal release
profile.
. Furthermore, the formulation and methods of manufacture render a composition
that is
conveniently compressible, but not excessively friable.
In one embodiment, the formulations used in the methods of the invention
comprise at least one sustained release agent (for purposes of the present
invention,
controlled release and sustained release may be used interchangeably), for
example, at
least one sustained release agonist of endothelial nitric oxide synthase
(e.g., an HMG-
CoA reductase inhibitor and/or a precursor of nitric oxide such as L-
arginine). In
3o another embodiment, the L-arginine is slowly released into the system of a
subject. The
slow release of L-arginine creates a phannacokinetic profile of L-arginine
within the
plasma that provides NOS with a substantially constant supply of L-arginine
needed for
the production of NO. The formulations can, therefore, slowly dissolve in vivo
and
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release a substantially uniform amount of L-arginine over a time period to be
therapeutically effective for a subject. In another embodiment, the HMG-CoA
reductase
inhibitor is slowly released into the system of the subject. In a further
embodiment, the
production of NO is substantially uniform over a prolonged period of time.
In another aspect of the present invention, a composition for the treatment of
vascular diseases (including, but not limited to, cardiovascular,
cerebrovascular,
peripheral vascular diseases and disorders), intermittent claudication,
critical limb
ischemia, and Alzheimer's Disease is provided in the form of food. Such
compositions
in the form of food may also be used to increase vasodilation, increase NO
production
to and lower cholesterol. Preferably, the food is in the form of a bar such as
a prescription
health bar. Use of food enables the provision of larger amounts of L-arginine
than could
be incorporated into a single tablet. The present invention provides a bar
that can
provide more than 1 gram of L-arginine as well as other agents, as desired. In
one
embodiment, the L-arginine is added as an immediate release formulation, e.g.,
15 immediate release granulars of L-arginine, to a food bar. In another
embodiment, the bar
includes a sustained release formulation that includes, e.g., sustained
release granulars of
L-arginine. In another embodiment, the bar further contains additional agents,
such as
an HMG-CoA reductase inhibitor. Preferably, the HMG-CoA reductase inhibitor,
is a
statin such as simvastatin.
2o
Definitions
Before further description of the invention, certain terms employed in the
specification, examples and claims are, for convenience, collected here.
As used herein, unless otherwise specified, the term "subject" includes
mammals.
25 The term "marmnals" includes, but is not limited to, dogs, cats, cattle,
horses, pigs, and
humans.
As used herein, the terms "treat", "treating", "treatment" and the like refer
to the
application or administration of a therapeutic agent or formulation to a
patient, or
application or administration of a therapeutic agent or formulation to an
isolated tissue
3o from a patient, who has a disease or disorder, a symptom of disease or
disorder or a
predisposition toward a disease or disorder, with the purpose of curing,
healing,
alleviating, relieving, altering, remedying, preventing, ameliorating,
delaying onset of the
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disease or disorder and/or event, slowing the progression of the disease or
disorder,
improving or affecting the disease or disorder, the symptoms of disease or
disorder or the
predisposition toward a disease or disorder and/or event.
As used herein,; the term "vascular disease" or "vascular disorder" generally
refer
to diseases or disorders of blood vessels and include, but are not limited to,
cardiovascular, cerebrovascular, and peripheral vascular diseases or
disorders.
Cardiovascular disease refers to diseases of blood vessels of the heart. See,
e.g., Kaplan,
R. M., et al., "Cardiovascular diseases" in Health and Human Behavior, pp. 206-
242
(McGraw-Hill, New York 1993). Cardiovascular disease is generally one of
several
l0 forms, including, for example, hypertension (also referred to as high blood
pressure),
coronary heart disease, stroke, and rheumatic heart disease. Peripheral
vascular disease
or disorders refer to diseases of any of the blood vessels outside of the
heart. For
example, peripheral vascular disease may refer to a narrowing of the blood
vessels that
carry blood to leg and arm muscles. Cerebrovascular disease refers to diseases
that
affect the ability of blood vessels to supply blood to the brain.
The term "atherosclerosis" encompasses vascular diseases and disorders and
conditions that are recognized and understood by physicians practicing in the
relevant
fields of medicine. Atherosclerotic cardiovascular disease, coronary heart
disease (also
known as coronary artery disease or ischemic heart disease), cerebrovascular
disease and
2o peripheral vessel disease are all clinical manifestations of
atherosclerosis and are
therefore encompassed by the terms "atherosclerosis" and "atherosclerotic
disease".
As used herein the terms "coadministration" or "coadministered" when used to
describe the administration of two or more compounds to a subject means that
the
compounds, which may be administered by the same or different routes, are
administered concurrently (e.g., as a mixture) or sequentially, such that the
pharmacological .effects of each overlap in time. As used herein, unless
otherwise
specified, when applied to the administration of at least two compounds, the
term
"sequentially" means that the compounds are administered such that the
pharmacological
effects of each overlap in time. In certain embodiments, agents are
coadministered
3o substantially simultaneously. By "substantially simultaneously," it is
meant that the
formulation of the invention is administered to the subject close enough in
time with the
administration of at least one additional agent, whereby the agents may exert
an additive
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or even synergistic effect, e.g., without limitation, increasing NOS activity,
NO
production, or vasodilation.
As used herein the term "precursor of NO" includes any substrate precursor of
native NO, e.g., L-arginine.
The term "native NO" as used herein refers to nitric oxide that is produced
through the bio-transformation of L-arginine or the L-arginine dependent
pathway. The
terms "endothelium derived relaxing factor (EDRF)" or "endothelium derived
nitric
oxide (EDNO)" may be used interchangeably with "native NO".
As used herein the term "L-arginine" refers to L-arginine and all of its
to biochemical equivalents, e.g., L-arginine hydrochloride, precursors, and
its basic form,
that act as substrates of NOS with resulting increase in production of NO. The
term
includes pharmaceutically acceptable salts of L-arginine.
The term "pharmaceutically acceptable salts" refers to salts prepared from
pharmaceutically acceptable non-toxic acids or bases including inorganic acids
and bases
and organic acids and bases. Suitable non-toxic acids include inorganic and
organic
acids such as acetic, benzenesulfonic, benzoic, camphorsulfonic, citric,
ethenesulfonic,
furnaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic,
malefic, malic,
mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric,
succinic,
sulfuric, tartaric acid, p-toluenesulfonic, and the like. Particularly
preferred are
hydrochloric, hydrobromic, phosphoric, and sulfuric acids, and most
particularly
preferred is the hydrochloride salt.
Since the L-arginine used in the methods of the present invention is both
basic
and acidic, salts may be prepared from pharmaceutically acceptable non-toxic
acids or
bases including inorganic and organic acids or inorganic and organic bases.
Such salts
may contain any of the following anions: acetate, benzensulfonate, benzoate,
camphorsulfonate, citrate, fumarate, gluconate, hydrobromide, hydrochloride,
lactate,
maleate, mandelate, mucate, nitrate, pamoate, phosphate, succinate, sulfate,
tartrate, and
the like. Particularly preferred are benzensulfonate, hydrobromate,
hydrochloride, and
sulfate. Such salts may also contain the following canons: aluminum, calcium,
lithium,
3o magnesitun, potassium, sodium, zinc, benzathine, chloroprocaine, choline,
diethanolamine, ethylenediamine, meglumine, and procaine.
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As used herein the term "agonist" or "agonist of eNOS or cNOS" refers to an
agent which stimulates the bio-transformation of a substrate such as, for
example, L-
arginine to NO. An agonist of eNOS or cNOS includes, for example, an HMG-CoA
reductase inhibitor. "HMG-CoA reductase (3-hydroxy-3-methylglutaryl-coenzyme
A)"
is the microsomal enzyme that catalyzes the rate limiting reaction in
cholesterol
biosynthesis. An "HMG-CoA reductase inhibitor" inhibits HMG-CoA reductase.
HMG-CoA reductase inhibitors are also referred to as "statins."
There are a large number of compounds described in the art that have been
obtained naturally or synthetically, which inhibit HMG-CoA reductase and are
referred
to as "statins," and which form the category of agents useful for practicing
the present
invention. Examples include, without limitation, those which are commercially
available, such as simvastatin (LT.S. Pat. No. 4,444,784), lovastatin (U.S.
Pat. No.
4,231,938), pravastatin sodium (LT.S. Pat. No. 4,346,227), fluvastatin (U.S.
Pat. No.
4,739,073), atorvastatin (U.S. Pat. No. 5,273,995), cerivastatin,
rosuvastatin, and
numerous others such as compactin, dalvastatin, mevastatin, fluindostatin,
pitavastatin,
HR-780, GR-95030, CI 980, BMY 22089, BMY 22566, and those described in, for
example, U.S. Pat. No. 5,622,985, U.S. Pat. No. 5,135,935, U.S. Pat. No.
5,356,896,
U.S. Pat. No. '4,920,109, U.S. Pat. No. 5,286,895, U.S. Pat. No. 5,262,435,
U.S. Pat. No.
5,260,332, U.S. Pat. No. 5,317,031, U.S. Pat. No. 5,283,256, U.S. Pat. No.
5,256,689,
2o U.S. Pat. No. 5,182,298, U.S. Pat. No. 5,369,125, U.S. Pat. No. 5,302,604,
U.S. Pat. No.
5,166,171, U.S. Pat. No. 5,202,327, U.S. Pat. No. 5,276,021, U.S. Pat. No.
5,196,440,
U.S. Pat. No. 5,091,386, U.S. Pat. No. 5,091,378, U.S. Pat. No. 4,904,646,
U.S. Pat. No.
5,385,932, U.S. Pat. No. 5,250,435, U.S. Pat. No. 5,132,312, U.S. Pat. No.
5,130,306,
U.S. Pat. No. 5,116,870, U.S. Pat. No. 5,112,857, U.S. Pat. No. 5,102,911,
U.S. Pat. No.
5,098,93 1, U.S. Pat. No. 5,081,136, U.S. Pat. No. 5,025,000, U.S. Pat. No.
5,021,453,
U.S. Pat. No. 5,017,716, U.S. Pat. No. 5,001,144, U.S. Pat. No. 5,001,128,
U.S. Pat. No.
4,997,837, U.S. Pat. No. 4,996,234, U.S. Pat. No. 4,994,494, U.S. Pat. No.
4,992,429,
U.S. Pat. No. 4,970,231, U.S. Pat. No. 4,968,693, U.S. Pat. No. 4,963,538,
U.S. Pat. No.
4,957,940, U.S. Pat. No. 4,950,675, U.S. Pat. No. 4,946,864, U.S. Pat. No.
4,946,860,
U:S. Pat. No. 4,940,800, U.S. Pat. No. 4,940,727, U.S. Pat. No. 4,939,143,
U.S. Pat. No.
4,929,620, U.S. Pat. No. 4,923,861, U.S. 'Pat. No. 4,906,657, U.S. Pat. No.
4,906,624
and U.S. Pat. No. 4,897,402, the disclosures of each of which are incorporated
herein by
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reference. Any other member of the class of compounds that inhibits HMG-CoA
reductase maybe used in the methods of the invention. A combination of two or
more
HMG-CoA reductase inhibitors may also be used in the methods of the invention.
The term "eNOS activity", as used herein, means the ability of a cell to
generate
NO from the substrate L-arginine. Increased eNOS activity can be accomplished
in a
number of different ways. For example, an increase in the amount of eNOS
protein or
an increase in the activity of the protein (while maintaining a constant level
of the
protein) can result in increased "activity." An increase in the amount of
protein available
can result from, for example and without limitation, increased transcription
of the eNOS
l0 gene, increased translation of eNOS mRNA, increased stability of the eNOS
mRNA,
activation of eNOS, or a decrease in eNOS protein degradation.
The eNOS activity in a cell or in a tissue can be measured in a variety of
different
ways. A direct measure is to measure the amount of eNOS present. Another
direct
measure is to measure the amount of conversion of L-arginine to L-citrulline
by eNOS or
the amount of nitric oxide generation by eNOS under particular conditions,
such as the
physiologic conditions of the tissue. The eNOS activity also can be measured
indirectly,
for example by measuring mRNA half life (an upstream indicator) or by a
phenotypic
response to the presence of NO (a downstream indicator). One phenotypic
measurement
employed in the art is measuring endothelial dependent relaxation in response
to
2o acetylcholine, which response is affected by eNOS activity. The level of NO
present in a ,
sample can be measured using a NO meter. All of the foregoing techniques are
well
known to those of ordinary skill in the art.
The methods of the present invention, by causing an increase in NO production,
permit not only the re-establishment of normal base-line levels of eNOS
activity, but
also allow increasing such activity above normal base-line levels. Normal base-
line
levels are the amounts of activity in a normal control group, controlled for
age and
having no symptoms that would indicate alteration of endothelial cell NOS
activity (such
as hypoxic conditions, hyperlipidemia and the like). The actual level then
will depend
upon the particular age group selected and the particular measure employed to
assess
3o activity. In abnormal circumstances, endothelial cell NOS activity (and NO
production)
is depressed below normal levels. Accordingly, the formulations of the
invention can
not only restore normal base-line levels of NO production in such abnormal
conditions,
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but can increase endothelial cell NOS activity (and NO production) far above
normal
base-line levels.
The teen "tamer" refers to diluents, excipients and the like for use in
preparing
admixtures of a pharmaceutical composition.
As used herein, the term "dosage form" means a pharmaceutical composition that
contains an appropriate amount of active ingredient for administration to a
subject, e.g.,
a patient either in single or multiple doses.
The unit "mglKg" as used herein means the mg of agent per I~g of subject body
weight.
to As used herein, unless otherwise indicated, the term "half life" means the
time
taken to decrease the concentration of drug in the blood plasma of the
organism by about
one half from the drug concentration at the time of administration.
As used herein, unless otherwise specified, the term "immediate release" means
that no extrinsic factors delay the ih vitro release of one or more drugs.
15 As used herein, the terms "pharmaceutical composition" or "pharmaceutical
formulation," used interchangeably herein, mean a composition that comprises
pharmaceutically acceptable constituents.
As used herein, the term "pharmaceutically acceptable" means the type of
formulation that would be reviewed and possibly approved by a regulatory
agency of the
20 Federal or a state government or listed in the U.S. Pharmacopeia or other
generally
recognized pharmacopeia for use in animals, and more particularly in humans.
As used herein unless otherwise specified, the term "pharmaceutically
acceptable
carrier" means a carrier medium which does not interfere with the
effectiveness of the
biological activity of the active ingredient and which is not toxic to the
subject to which
25 it is administered. The use of such media and agents for pharmaceutically
active
formulations is well known in the art. Except insofar as any conventional
media or agent
is incompatible with the active compound, use thereof in the formulations used
in the
methods of the invention is contemplated.
As used herein, the term "pharmaceutically acceptable salts" refers to salts
3o prepared from pharmaceutically acceptable non-toxic acids, including
inorganic acids
and organic acids.
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As used herein, unless otherwise specified, the term "sustained release" is
defined as a prolonged release pattern of one or more drugs, such that the
drugs are
released over a period of time. For purposes of the present invention,
sustained release
and controlled release are used interchangeably.
As used herein, the term "salt or complex" is used to describe a compound or
composition comprising two or more chemical moieties that are associated by at
least
one type of interaction including, but not limited to, Van der Waals, ionic
and/or
hydrogen bonding. A salt or complex may exist as a solid or in a liquid.
As used herein, the term "weight percent" when used to describe the amount of
a
l0 component within a formulation means the weight of the specified component
based
upon the weight of all components within the formulation.
Various aspects of the invention are described in further detail in the
following
subsections:
15 I. Formulations Used In Methods of Treatment or Prevention of
Cerebrovascular
and Cardiovascular Diseases and Disorders
The methods of the invention include methods of treating and preventing
cerebrovascular and/or cardiovascular diseases or disorders in a subject,
e.g., a human,
comprising administering to the subject a formulation comprising an HMG-CoA
2o reductase inhibitor and a formulation comprising L-arginine, either
concurrently or
sequentially. Alternatively, a single formulation comprising L-arginine and an
HMG-
CoA reductase inhibitor is administered to a subject.
One embodiment of the invention encompasses formulations comprising L-
arginine in a sustained release formulation, an HMG-CoA reductase inhibitor in
a
25 sustained release formulation, or both L-arginine and an HMG-CoA reductase
inhibitor
in a sustained release formulation. In one embodiment, the invention
encompasses
formulations comprising L-arginine that may be administered either
concurrently or
sequentially with at least one HMG=CoA reductase inhibitor wherein the
formulation
releases L-arginine in a substantially constant concentration over a prolonged
period of
30 time and the HMG-CoA reductase inhibitor is present in an immediate release
formulation. In another embodiment, the invention encompasses formulations
comprising L-arginine in a high concentration and in a sustained release
formulation
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wherein the pharmacokinetic profile is zero order release kinetics (i. e.,
linear release rate
over time). The release characteristics of both classes of drugs may be
modified to
provide release patterns that allow for the adaptation of the combination into
a once- daily
single unit dosage.
In one embodiment, the formulations used in the methods of the invention
comprise L-arginine in a therapeutically effective amount, an HMG-CoA
reductase
inlubitor in a therapeutically effective amount, and at least one sustained
release agent.
The formulations also can include additional ingredients necessary to modify
the
formulations for administration, preservation, esthetics and the like. In one
embodiment,
to the formulation of the present invention also include binders, fillers and
lubricants. In a
preferred embodiment, the formulation comprises a sustained release L-arginine
formula
comprising L-axginine, a binder, one or more sustained release agents, a
glidant, and a
release agent or lubricant. The formulation may further comprise fillers
and/or
compression agents. The sustained release formulations of the present
invention are
particularly advantageous because their release profile allows the
administration of lower
dosages to maintain the same level of drug in the body than required with
immediate
release or commercially available sustained release agents. Because
administration of
the sustained release L-arginine with a statin can also increase the
effectiveness of the
statin, e.g., simvastatin, the use of the formulations of the invention may
also allow a
lower dosage of statin with an equivalent beneficial affect.
L-arginine is commercially available from a number of sources known to the
skilled practitioner. USP grade L-arginine, for example, is commercially
available from
various sources including Sigma-Aldrich (Milwaukee, Wl). Suitable arginine and
arginine derivative compounds include, but are not limited to, arginine salts
such as
arginine HCl, arginine aspartate, or arginine nicotinate. Other arginine
compounds or
derivatives may be chosen from di-peptides that include arginine such as
alanylarginine
(ALA-ARG), valinyL-arginine (VAL-ARG), isoleucinyL-arginine (ISO-ARG), and
leucinyL-arginine (LEU-ARG), and tri-peptides that include arginine such as
argininyl-
lysinyl-glutamic acid (ARG-LYS-GLU) and arginyl-glysyL-arginine (ARG-GLY-ARG).
3o The L-arginine preferably is L-arginine monohydrochloride.
W one embodiment, the L-arginine is present at about 10% to about 90% by
weight of the formulation. In another embodiment, the L-arginine is present at
about
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25% to about 75% by weight of the formulation. In various embodiments, the L-
arginine is present at about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89
or 90%. In particular embodiments, the L-arginine is present at about 50, 51,
56, 69 or
70%. A~1 ranges within each of the above ranges are within the scope of the
present
invention.
Use of one or more sustained release agents allows for the slow release of the
L-
arginine andlor the HMG-CoA reductase inhibitor over an extended period of
time. For
example, the sustained release agent may release L-arginine at a rate that
will not cause
to concentration peaks or lows that would exacerbate side effects associated
with high or
low concentrations of L-arginine within the bloodstream. Sustained release
agents
suitable for the formulations used in the methods of the present invention
include
hydration agents, e.g., such as cellulose, that partially hydrate when in
contact with an
aqueous environment to form a gelatinous barner that retards dissolution of
the agent
that the hydration agent is coating. In other words, the sustained release
agents form a
temporary barrier to water such that water is slowly absorbed into the
formulation
thereby hydrating the formulation and subsequently releasing the active
ingredient, e.g.,
L-arginine, at a rate substantially slower than a formulation without
sustained release
agents. Additionally, the sustained release agents are present in a particle
size where
2o upon incorporation into a capsule or compaction or compression into a
tablet, pill, or
gelcap water slowly permeates into the structure.
Iti one embodiment, the sustained release agent or agents include, but are not
limited '~to, cellulose ether products, polymethylmethacrylate, or
polyvinylalcohol. In
another embodiment, sustained release agents include celluloses including, but
not
limited to methylcellulose, hydroxypropyl methylcellulose,
hydroxyethylcelluhose, or
combinations thereof. In a preferred embodiment, the sustained release agents
include
one or more hydroxypropyl methylcelluloses. Suitable sustained release agents
are
commercially available from The Dow Chemical Company under the trade
designations
METHOCEL~ and ETHOCEL~. In a preferred embodiment, the sustained release
3o agent is METHOCEL~ K100 M CR Premium and/or METHOCEL~ E 4M CR
Premium.
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The sustained release agent is typically present in an amount sufficient to
release
the active ingredient, e.g., L-arginine or an HMG-CoA reductase inhibitor,
over a desired
period of time. In one embodiment, the sustained release agent is present in
an amount
of about 5% to about 40% by weight of the formulation. hl another embodiment,
the
sustained release agent is present in an amount of about 5% to about 75% by
weight. In
yet another embodiment, the sustained release agent is present in an amount of
about
15% to about 50% by weight of the formulation. In various embodiments, the
sustained
release agent is present at about 5% to about 40%, for example, about 24% to
about
25%, about 27% to about 28%, about 31% to about 32%, and about 35%. In
alternative
to embodiments, the sustained release agent is present at about 40% to about
60%, for
example, about 45%. All ranges within each of the above ranges are within the
scope of
the present invention.
In one embodiment, the sustained release agent releases L-arginine over a
period
of 10 hours, as depicted in Figure 1. In one embodiment, the formulation
releases L-
arginine substantially uniformly over a period from about 4 hours to about 24
hours. In
another embodiment, the formulation of the present invention releases L-
arginine
substantially uniformly over a period of about 8 hours to about 24 hours. In
yet another
embodiment, the sustained release L-arginine formulation releases L-arginine
substantially uniformly over a period of about 12 hours to about 48 hours.
2o In another embodiment, a formulation used in the methods of the present
invention will release L-arginine in a manner to provide a pharmacokinetic
profile
wherein the half life (Tlia) and the Tmax are sufficient to maintain L-
arginine at a
substantially constant level. In other words, in one embodiment, a sustained
release
formulation of the invention releases L-axginine such that a steady state of
circulating L-
arginine is achieved and remains constant. In one embodiment, the
phaxmacolcinetic
profile is such that Tli2 is from about 4 hours to about 12 hours and the TmaX
is about 4
hours. In yet another embodiment, Tli2 is from about 4 hours to about 8 hours
and the
Tmax is about 4 hours.
Binders useful in the formulation include those commonly known to the skilled
3o practitioner. Binders include, but are not limited to, sugars, such as
lactose, sucrose,
glucose, dextrose, and molasses; natural and synthetic gums, such as acacia,
guar gum,
sodium alginate, extract of Irish moss, panwar gum, ghatti gum; other binders
include a
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mixture of polyethylene oxide and polyethylene glycol, methylcellulose, sodium
carboxymethylcellulose, hydroxypropyl cellulose (HPC), hydroxyethyl cellulose,
hydroxypropyl methylcellulose, alginic acid, ethyl cellulose, microcrystalline
cellulose,
carbomer, zero, starch, dextrin, maltodextrin, gelatin, pregelatinized starch,
polyvinlypyrrolidone (PVP) or povidone, and mixtures thereof. In a preferred
embodiment, the binder is polyvinylpyrrolidone homopolymer.
hi one embodiment, the binder is present at less than about 20% by weight of
the
formulation. In various embodiments, the binder is present at about .5% to
about 10%,
for example, about .5% to about 5%, about 2% to about 3%, about 3% to about
4%,
to about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7%
to about
8%, about 8% to about 9%, or about 9% to about 10%. All ranges within each of
the
above ranges are within the scope of the present invention.
In a preferred embodiment, the formulation of sustained release L-arginine
also
includes a glidant. The glidant can be any known USP grade glidant including,
e.g.,
silicon dioxide. In a preferred embodiment, the glidant is colloidal silicone
dioxide.
In one embodiment, the glidant is present at less than about 3% by weight of
the
formulation. In another embodiment, the glidant is present at less than about
2% of the
formulation. In a preferred embodiment, the glidant is present at less than
about 1 % by
weight of the formulation.
2o Fillers useful in the formulation include those commonly known to the
skilled
artisan. Typical fillers include, but are not limited to, sugars such as
lactose, sucrose,
dextrose, mannitol, and sorbitol, whey, dibasic calcium phosphate, tribasic
calcium
phosphate, calcium sulfate, and mixtures thereof. Other fillers include, but
are not
limited to, cellulose preparations such as maize starch, wheat starch, rice
starch, potato
starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropyl
methylcellulose,
sodium carboxymethylcellulose, polyvinylpyrrolidone, and mixtures thereof.
Microcrystalline cellulose can also function as a compression agent as well as
a filler. In
a preferred embodiment the fillerlcompression agent is microcrystalline
cellulose. More
preferably, the microcrystalline cellulose is that sold under the designation
AVICEL~
3o PH 102 by The Dow Chemical Company.
In one embodiment, the filler is present at less than about 50% by weight of
the
formulation. In another embodiment, the filler is present at about 2% to about
20% by
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weight of the formulation including, for example, at about 8% to about 9%, at
about 9%
to about 10%, at about 10% to about 11 %, at about 11 % to about 12%, and at
about 12%
to about 13% by weight of the formulation. In a preferred embodiment, the
filler is
present at about 10% by weight of the formulation. All ranges within each of
the above
ranges are within the scope of the present invention.
Excipients can be added to increase the amount of solids present in the
formulation. Among the excipients found useful for this purpose, often in
combination,
are sodium or potassium phosphates, calcium carbonate, calcium phosphate,
sodium
chloride, citric acid, tartaric acid, gelatin, and carbohydrates such as
dextrose, sucrose,
l0 lactose, sorbitol, inositol, mannitol and dextran, starches, cellulose
derivatives, gelatin,
and polymers such as polyethylene glycols. In addition to those mentioned
herein, others
are known to those skilled in the art.
Release agents or lubricants useful in the formulation include those commonly
known to the skilled artisan. Typical lubricants include, but are not limited
to, stearate,
magnesium stearate, zinc stearate, calcium stearate, stearic acid,
hydrogenated vegetable
oils (e.g., hydrogenated cottonseed oil), sodium stearyl fumarate, glyceryl
palmitostearate, glyceryl behenate, sodiiun benzoate, sodium lauryl sulfate,
magnesium
lauryl sulfate, mineral oil, talc, and mixtures thereof. In a preferred
embodiment, the
lubricant is magnesium stearate. In other embodiments, lubricants are chosen
so as to
insure optimal absorption and utilization of nutrients.
In one embodiment, the lubricant is present at less than about 20% by weight
of
the formulation. In another embodiment, the lubricant is present at about 2%
to about
20% by weight of the formulation. In a preferred embodiment, the.lubricant is
present at
about 10% by weight of the formulation.
Disintegrants include, but are not limited to, sodium starch glycolate,
croscarmellose sodium, crospovidone, cross-linked polyvinylpyrrolidone, corn
starch,
pregelatinized starch, microcrystalline cellulose, alginic acid, amberlite ion
exchange
resins, polyvinylpyrrolidone, polysaccharides, sodium carboxymethylcellulose,
agar,
salts thereof such as sodium alginate, Primogel, and mixtures thereof.
The compression agent allows for the formulation to be shaped into a tablet,
troche, gelcap, or other presentation for administration in solid form. In one
embodiment, the compression agent allows the formulation to be shaped into a
tablet,
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troche, or gelcap. Compression agents include, but are not limited to, Avicel,
magnesium stearate, wax, gums, celleusics, stearate, or combinations thereof.
In a
preferred embodiment, the compression agent is microcrystalline cellulose.
In one embodiment, the compression agent is present in an amount of about
0.01% to about 5% by weight percent of the formulation. In another embodiment,
the
compression agent is present in an amount of about 0.5% to about 3%. In yet
another
embodiment, the compression agent is present in an amount of about 1 % to
about 2% by
weight of the formulation.
In one embodiment, the L-arginine formula includes L-arginine in a unit dosage
to that would be sufficient for about 5 mg/I~g to about 40 mg/Kg subject body
weight. fii
another embodiment, the L-arginine formula includes L-arginine in a unit
dosage that
would be sufficient for about 20 mg/Kg to about 25 mg/Kg.
In another embodiment, both L-arginine and am HMG-CoA reductase inhibitor
are in a sustained release formulation. The amount of HMG-CoA reductase
inhibitor
may vary based on the specific inhibitor present in the formulation, as some
inhibitors
are more efficacious than others. For example, BAYCOL~ may be present in an
amount
of about 0.1 mg to about 0.8 mg per tablet, and ZOCOR~ may be present in an
amount
of about 10 mg to about 80 mg.per tablet. Those skilled in the art will be
able to
determine a therapeutic amount based on the specific inhibitor employed. In
one
2o embodiment, the HMG-CoA reductase inhibitor is simvastatin and is present
in a unit
dosage that would be sufficient for about 0.5 mg/Kg to about 3 mg/Kg subject
body
weight. In another embodiment, the HMG-CoA reductase inhibitor is simvastatin
and is
present in a unit dosage that would be sufficient for about 1.2 mg/Kg to about
1.4 mg/I~g
subject body weight.
In yet another embodiment, the L-arginine and HMG-CoA reductase inhibitor are
both provided in separate sustained release formulations, e.g., separate
tablets.
Sustained release HMG-CoA reductase inhibitor is commercially available from,
e.g.,
Merck & Company, Inc. (Rahway, NJ).
Formulations used in the methods of the invention may comprise a
3o pharmaceutical carrier according to conventional pharmaceutical compounding
techniques. The carrier may take a wide variety of forms depending on the form
of the
preparation desired for oral administration. In preparing the formulations for
oral dosage
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form any of the usual pharmaceutical media may be employed. The most preferred
oral
solid preparations are tablets and gelcaps. Alternatively, the formulations of
the present
invention may be incorporated into a capsule. In this embodiment, the
sustained release
L-arginine granulars, and, optionally, the HMG-CoA reductase inhibitor, may be
incorporated within a capsule.
Because of their ease of administration, tablets and capsules represent the
most
advantageous oral dosage unit form, in which case solid pharmaceutical
carriers are
employed. Tablets or capsules may contain an L-arginine formulation and HMG-
CoA
reductase inhibitor formulation in the same tablet or capsule in different
configurations.
1o Configurations include, a two-part half and half tablet or capsule, one
formulation
surrounding a second, dispersion of one formulation in another, granules of
both
formulations intermixed, and the like. If desired, tablets or capsules may be
coated by
standard aqueous or non-aqueous techniques.
The formulations used in the methods of the present invention may also
comprise
other pharmaceutically acceptable ingredients, such as those commonly used in
the art.
See, Remington: the Science & Practice of Pharmacy, by Alfonso R. Gennaro,
20th ed.,
Williams & Wilkins, 2000. Additional ingredients used in the formulations used
in the
methods of the present invention include, but are not limited to, water,
glycols, oils,
alcohols, starches, sugars, diluents, disintegrating agents, preservatives,
excipients,
lubricants, disintegrants, diluents, Garners, stabilizing agents, coloring
agents, flavoring
agents, and combinations thereof. Examples of suitable diluents include water,
ethanol,
polyols, vegetable oils, injectable organic esters such as ethyl oleate, and
combinations
thereof. Formulations can also contain adjuvants such as preserving, wetting,
emulsifying, and dispensing agents. Prevention of the action of microorganisms
can be
insured by various antibacterial and antifungal agents including, but not
limited to,
parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be
desirable to
include isotonic agents including, but not limited to, sugars, sodium
chloride, and the
like.
In another embodiment of the invention, the formulations may be further
3o co-administered with at least one other pharmaceutical agent. Examples of
categories of
pharmaceutical agents include: adrenergic agent; adrenocortical steroid;
adrenocortical
suppressant; aldosterone antagonist; amino acid; ammonia detoxicant; anabolic;
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analeptic; analgesic; androgen; anesthetic; anorectic; antagonist; anterior
pituitary
suppressant; anthelinintic; anti-acne agent; anti-adrenergic; anti-allergic;
anti-amebic;
anti-androgen; anti-anemic; anti-anginal; anti-anxiety; anti-arthritic; anti-
asthmatic; anti-
atherosclerotic; antibacterial; anticholelithic; anticholelithogenic;
anticholinergic;
anticoagulant; anticoccidal; anticonvulsant; antidepressant; antidiabetic;
antidiarrheal;
antidiuretic; anti-emetic; anti-epileptic; anti-estrogen; antifibrinolytic;
antifungal;
antiglaucoma agent; antihemophilic; antihemorrhagic; antihistamine;
antihyperlipidemia;
antihyperlipoproteinemic; antihypertensive; anti-infective; anti-inflammatory;
antikeratinizing agent; antimalarial; antimicrobial; antimigraine;
antimitotic;
antimycotic, antinauseant, antineoplastic, antineutropenic, antiobessional
agent;
antiparasitic; antiparkinsonian; antiperistaltic, antipneumocystic;
antiproliferative;
antiprostatic hypertrophy; antiprotozoal; antipruritic; antipsychotic;
antirheumatic;
antischistosomal; antiseborrheic; antisecretory; antispasmodic;
antithrombotic;
antitussive; anti-ulcerative; anti-urolithic; antiviral; appetite suppressant;
benign
prostatic hyperplasia therapy agent; blood glucose regulator; bone resorption
inhibitor;
bronchodilator; carbonic anhydrase inhibitor; cardiac depressant;
cardioprotectant;
cardiotonic; cardiovascular agent; choleretic; cholinergic; cholinesterase
deactivator;
coccidiostat; cognition adjuvant; depressant; diuretic; dopaminergic agent;
ectoparasiticide; emetic; enzyme inhibitor; estrogen; fibrinolytic;
fluorescent agent; free
oxygen radical scavenger; gastrointestinal motility effector; glucocorticoid;
gonad-
stimulating principle; hair growth stimulant; hemostatic; histamine H2
receptor
antagonists; hormone; hypocholesterolemic; hypoglycemic; hypolipidemic;
hypotensive;
imaging agent; immunizing agent; immunomodulator; immunoregulator;
immunostimulant; immunosuppressant; impotence therapy adjunct; keratolytic;
LNRII
agonist; liver disorder treatment; luteolysin; mental performance enhancer;
mood
regulator; mucolytic; mucosal protective agent; mydriatic; nasal decongestant;
neuromuscular blocking agent; neuroprotective; NMDA antagonist; non-hormonal
sterol
derivative; oxytocic; plasminogen activator; platelet activating factor
antagonist; platelet
aggregation inhibitor; potentiator; progestin; prostaglandin; prostate growth
inhibitor;
3o prothyrotropin; psychotropic; radioactive agent; regulator; relaxant;
repartitioning agent;
scabicide; sclerosing agent; sedative; selective adenosine Al antagonist;
serotonin
antagonist; serotonin inhibitor; serotonin receptor antagonist; steroid;
stimulant;
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suppressant; symptomatic multiple sclerosis; synergist; thyroid hormone;
thyroid
inhibitor; thyromimetic; tranquilizer; treatment of cerebral ischemia;
treatment of Paget's
disease; treatment of unstable angina; uricosuric; vasoconstrictor;
vasodilator; vulnerary;
wound healing agent; or xanthine oxidase inhibitor.
Another example of a pharmaceutical agent includes angiotensin converting
enzyme inhibitors (ACE inhibitors). ACE is an enzyme that catalyzes the
conversion of
angiotensin I to angiotensin II. ACE inhibitors include amino acids and
derivatives
thereof, peptides, including di and tri peptides and antibodies to ACE which
intervene in
the renin-angiotensin system by inhibiting the activity of ACE thereby
reducing or
to eliminating the formation of pressor substance angiotensin II. ACE
inhibitors have been
used medically to treat hypertension, congestive heart failure, myocardial
infarction and
renal disease. Classes of compounds known to be useful as ACE inhibitors
include
acylinercapto and mercaptoalkanoyl prolines such as captopril (U.S. Pat. No.
4,105,776)
and zofenopril (U.S. Pat. No. 4,316,906), carboxyalkyl dipeptides such as
enalapril (U.S.
15 Pat. No. 4,374,829), lisinopril (U.S. Pat. No. 4,374,829), quinapril (U.S.
Pat. No.
4,344,949), ramipril (US Pat. No. 4,587,258), and perindopril (U.S. Pat. No.
4,508,729),
carboxyalkyl dipeptide mimics such as cilazapril (U.S. Pat. No. 4,512,924) and
benazapril (LT.S. Pat. No. 4,410,520), phosphinylalkanoyl prolines such as
fosinopril
(U.S. Pat. No. 4,337,201) and trandolopril. Estrogens upregulate NOS
expression
20 whereas ACE inhibitors do not affect expression, but instead influence the
efficiency of
the action of NOS on L-arginine. Thus, activity can be increased in a variety
of ways. In
general, activity is increased by the reductase inhibitors of the invention by
increasing
the amount of the active enzyme present in a cell versus the amount present in
a cell
absent treatment with the reductase inhibitors according to the invention.
II. Prophylactic and Ther~eutic Methods
In one aspect, the invention provides methods for preventing vascular diseases
or
disorders, such as cerebrovascular and/or cardiovascular diseases or
disorders, in a
subject by administering to a subject at risk for cerebrovascular and/or
cardiovascular
diseases or disorders a formulation comprising L-arginine along with a
formulation
comprising an HMG-CoA reductase inhibitor (e.g., simvastatin), either
sequentially, or
concurrently, or a single formulation comprising L-arginine along with an HMG-
CoA
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reductase inhibitor. Subjects at risk for cerebrovascular and/or
cardiovascular diseases
and disorders (including events) can be identified by, for example, a
predisposition to
atherosclerosis, symptoms of atherosclerosis, or by the presence of risk
factors such as,
for example, cigarette smoking, high blood pressure, diabetes, family history,
genetic
factors, high cholesterol levels, advancing age and alcohol use.
Administration of a formulation used in the methods of the invention as a
prophylactic agent can occur prior to the manifestation of symptoms
characteristic of the
onset of cerebrovascular and/or cardiovascular disease or disorder, such that
cerebrovascular and/or cardiovascular disease or disorder is prevented, its
progression
to slowed, or its onset delayed.
As described in International Patent Publication No. WO 00/56403 entitled
"Upregulation of Type III Endothelial Cell Nitric Oxide Synthase By HMG-CoA
Reductase Inhibitors," incorporated in its entirety by this reference,
upregulation of NOS
activity does not depend upon a decrease in cholesterol synthesis and in
particular does
not depend upon a decrease in the formation of ox-LDL. The present invention,
therefore, is useful whenever it is desirable to restore eNOS activity or
increase such
activity in an affected cell or tissue. The tissue is defined as to include
both the cells in
the vasculature supplying nutrients to the tissue, as well as cells of the
tissue that express
eNOS.
2o Nitric Oxide Synthase activity is involved in many conditions, including
impotence, heart failure, gastric and esophageal motility disorders, kidney
disorders such
as kidney hypertension and progressive renal disease, insulin deficiency, etc.
Individuals
with such conditions may benefit from increased NO production. For example,
individuals with pulmonary hypertension often have reduced levels of Nitric
Oxide
Synthase expression in their pulmonary vessels and benefit clinically from
inhalation of
Nitric Oxide. The invention therefore is particularly useful for treating
pulmonary
hypertension. It also has been demonstrated that hypoxia causes an inhibition
of eNOS
activity. The invention therefore is useful for treating subjects with hypoxia-
induced
conditions. It also has been discovered, surprisingly, that HMG-CoA reductase
inhibitors are useful for reducing ID brain injury that occurs following a
stroke.
The subject can have a condition characterized by an abnormally low level of
eNOS activity which is hypoxia-induced. In other embodiments, the subj ect can
have a
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condition comprising an abnormally low level of eNOS activity that is
chemically
induced. In still other embodiments the subj ect can have a condition
comprising an
abnormally low level of eNOS activity that is cytokine induced. In certain
important
embodiments, the subject has pulmonary hypertension or an abnormally elevated
risk of
pulmonary hypertension. In other important embodiments, the subject has
experienced
an ischemic stroke or has an abnormally elevated risk of an ischemic stroke.
In still
other important embodiments, the subject has heart failure or progressive
renal disease.
In yet other important embodiments, the subject is chronically exposed to
hypoxic
conditions.
l0 In further important embodiments, the subject has experienced a thrombotic
event or has an abnormally elevated risk of thrombosis. In still other
embodiments, the
subject has an abnormally elevated risk of arteriosclerosis or has
arteriosclerosis. In
other important embodiments, the subject has an abnormally elevated risk of
developing
a myocardial infarction or has experienced a myocardial-infarction. In yet
another
embodiment, the subject has an abnormally elevated risk of reperfusion injury.
In
preferred embodiments, the subject with an elevated risk of reperfusion injury
is an
organ transplant recipient (e.g., heart, kidney, liver, etc.). In other
important
embodiments, the subject has homocystinuria. In certain other important
embodiments,
the subject has cerebral autosonial dominant arteriopathy with subcortical
infarcts and
leucoencephalopathy (CADASIL) syndrome. In further important embodiments, the
subject has a degenerative disorder of the nervous system. In preferred
embodiments,
the subject with a degenerative disorder of the nervous system has Alzheimer's
disease.
In certain other embodiments, when the subject in need of a treatment
according
to the present invention has an abnormally elevated risk of an ischemic
stroke, HMG-
CoA reductase inhibitors are excluded as treatments for such subjects.
In other embodiments, the methods and compositions (e.g., L-arginine sustained
release formulations, L-arginine food bars, etc.) of the present invention may
be used to
treat or prevent Alzheimer's Disease. In yet another embodiment, the methods
and
compositions of the present invention may be used to treat or prevent
intermittent
3o claudication. In yet another embodiment, the formulations and compositions
of the
present invention may be used to increase vasodilation.
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In a preferred embodiment, the methods of the present invention may be used to
lower cholesterol levels in a subject. Administering HMG-CoA reductase
inhibitor and
L-arginine to a subject can serve to lower total cholesterol. In one
embodiment, the
method lowers total cholesterol by about 50 to about 150 mg/dL. In another
embodiment, the method reduces total cholesterol by about 80 to about 100
mg/dL. In
addition, administering HMG-CoA reductase inhibitor and L-arginine to a
subject can
serve to lower low density lipoprotein (LDL) cholesterol. In one embodiment,
the
method lowers LDL cholesterol by about 40 to about 110 mg/dL. In another
embodiment the method lowers LDL cholesterol by about 60 to about 100 mg/dL.
The
to methods of the present invention may also serve to increase high density
lipoprotein
(HDL) cholesterol in a subj ect. Furthermore, the administration of HMG-CoA
reductase
inhibitor and L-arginine can lower triglycerides in a subject. In one
embodiment, the
methods of the invention lower triglycerides in a subject by about 30 to about
100
mg/dL. In another embodiment, the methods of the invention lower triglycerides
by
about 45 to about 75 mgldL.
The coadministration of HMG-CoA reductase inhibitor and L-arginine has a
synergistic effect in reducing cholesterol levels in a subj ect. The methods
and
compositions of the present invention have been shown to reduce cholesterol
levels at a
surprising and significant amount over other known methods and compositions.
In
particular, the coadministration of HMG-CoA reductase inhibitor and sustained
release
L-arginine in accordance with the present invention reduces triglycerides and
LDL levels
in a significant manner over preexisting methods. Moreover, the
coadministration of
HMG-CoA reductase inhibitor and sustained release L-arginine increase HDL in a
significant manner over preexisting methods. In one embodiment, the
coadministration
of HMG-CoA reductase and L-axginine lowers total cholesterol by about 5% to
about
15% more compared to administration of HMG-CoA reductase inhibitor alone. In
another embodiment, the coadministration of HMG-CoA reductase and L-arginine
lowers total cholesterol by about 5 to about 20 mg/dL more compared to
administration
of HMG-CoA reductase inhibitor alone. In yet another embodiment, the
3o coadministration of HMG-CoA reductase and L-arginine lowers LDL cholesterol
by
about 2 to about 20 mg/dL more compared to administration of HMG-CoA reductase
inhibitor alone. In yet another embodiment, the coadministration of HMG-CoA
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reductase and L-arginine lowers triglycerides by about 5 to about 50 mg/dL, or
alternatively by about 20 to about 35 mg/dL, more compared to administration
of HMG-
CoA reductase inhibitor alone.
In another embodiment, the methods of the present invention may be used to
lower C-reactive protein in a subject. C-reactive protein is an acute phase
reactant
released by the body in response to acute injury, infection, or other
inflammatory stimuli.
Studies have demonstrated a positive correlation between C-reactive protein
and
coronary artery disease. Ridker, Circulation 108(12): e81-85 (2003); Blake et
al., Am. J.
Physiol. Regul. Integr. Comp. Physiol. 285(5): 81250-1252 (2003). In one
embodiment,
l0 the methods lower C-reactive protein by about 10% to about 50%, or by about
25% to
about 35%.
The coadministration of HMG-CoA reductase inhibitor and L-arginine has a
synergistic effect in lowering C-reactive protein. In one embodiment, the
method lowers
C-reactive protein by about 50% to about 90%, or about 65% to about 75%, more
compared to administration of HMG-CoA reductase inhibitor without the
sustained
release formulation of L-arginine. In another embodiment, the method lowers C-
reactive
protein by about 80% to about 120%, or about 95% to about 105%, more compared
to
administration of the sustained release formulation of L-arginine without HMG-
CoA
reductase inhibitor.
Furthermore, methods of the present invention may be used to increase nitric
oxide production and/or increase vasodilation in a subj ect with elevated
asymmetrical
dimethylarginine (ADMA). Asymmetrical dimethylargiune (ADMA) is an endogenous,
competitive inhibitor of eNOS. The presence of elevated plasma ADMA levels is
associated with endothelial dysfunction. Statins stimulate the expression of
endothelial
NO synthase (eNOS) in vitro and enhance endothelium-dependent, NO-mediated
vasodilation in vivo. Accordingly, statins (e.g., simvastatin) can enhance
endothelial
fiulction in patients with elevated ADMA. Without wishing to be bound by
theory, it is
believed that the inhibitory effect of ADMA is overcome by L-arginine.
By administering L-arginine, and, optionally, an HMG-CoA reductase inhibitor
(e.g., simvastatin), to a subject with elevated ADMA, the methods of the
present
invention can increase nitric oxide production and/or increase vasodilation.
Such
coadministration can increase endothelial function by about 5% to about 15% or
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alternatively, by about 7% to about 12%. In one embodiment according to the
invention,
the subject has endothelial dysfunction.
For any mode of administration, the actual amount of compound delivered, as
well as the dosing schedule necessary to achieve the advantageous
pharmacokinetic
profiles described herein, will depend, in part, on such factors as the
bioavailability of
the compound (and/or an active metabolite thereof), the disorder being
treated, the
desired therapeutic dose, and other factors that will be apparent to those of
skill in the
art. The actual amount delivered and dosing schedule can be readily determined
by
those of skill without undue experimentation by monitoring the blood plasma
levels of
to administered compound and/or an active metabolite thereof, and adjusting
the dosage or
dosing schedule as necessary to achieve the desired pharmacokinetic profile.
The formulations used in the methods of the invention, as described herein, or
pharmaceutically acceptable addition salts or hydrates thereof, can be
delivered to a
subject so as to avoid or reduce undesirable side effects according to the
invention using
15 a wide variety of routes or modes of administration. In one embodiment, the
subj ect is
an animal. In another embodiment, the subject is a mammal. In yet another
embodiment, the subject is a human. The most suitable route in any given case
will
depend on the nature and severity of the condition being treated. The
preferred route of
administration of the present invention is the oral route. The compositions
may be
20 conveniently presented in unit dosage form, and prepared by any of the
methods well
known in the art of pharmacy. Techniques and formulations for administering
the
compositions may be found in Remington: the Science & Practice of Pharmacy, by
Alfonso R. Gennaro, 20th ed., Williams & Wilkins, 2000.
The formulations of the invention will generally be used in an amount
effective
25 to achieve the intended purpose, e.g., to treat and/or prevent a
cerebrovascular and/or
cardiovascular disease or disorder. By therapeutically effective amount is
meant an
amount effective to treat a disease, disorder, symptom related to a disease or
disorder, or
predisposition toward a disease or disorder. As described earlier, the term
"treat" refers
to the application or administration of a therapeutic agent or formulation to
a patient, or
3o application or administration of a therapeutic agent or formulation to an
isolated tissue
from a patient, who has a disease or disorder, a symptom of disease or
disorder or a
predisposition toward a disease or disorder, with the purpose of curing,
healing,
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alleviating, relieving, altering, remedying, ameliorating, delaying onset of
the disease or
disorder and/or event, slowing the progression of the disease or disorder,
improving or
affecting the disease or disorder, the symptoms of disease or disorder or the
predisposition toward a disease or disorder and/or event. Determination of a
therapeutically effective amount is well within the capabilities of those
skilled in that art,
especially in light of the detailed disclosure provided herein.
Pharmaceutical formulations suitable for use with the present invention
include
formulations wherein L-arginine and/or an HMG-CoA reductase inhibitor are
contained
in a therapeutically effective amount, i.e., an amount effective to achieve
the intended
1o purpose. In general, an effective amount is that amount of a pharmaceutical
preparation
that alone, or together with further doses, produces the desired response.
This may
involve only slowing the progression of the disease temporarily. In another
embodiment,
it involves halting the progression of the disease permanently or delaying the
onset of or
preventing the disease or condition from occurnng. The effect of the dosage on
any
i5 particular disease can be monitored by routine methods. Such amounts will
depend, of
course, on the particular condition being treated, the severity of the
condition, the
individual patient parameters including age, physical condition, size and
weight, the
duration of the treatment, the nature of concurrent therapy (if any), the
specific route of
administration and like factors within the knowledge and expertise of the
health
2o practitioner.
Generally, doses of active compounds would be from about 0.01 mg/kg per day
to about 1000 mg/kg per day. In one embodiment, it is expected that doses
ranging from
about 50 to about 500 mg/kg will be suitable. In another embodiment,
administration is
oral and in one or several administrations per day.
25 Of course, the actual amount of L-arginine and/or an HMG-CoA reductase
inhibitor will depend on, among other things, the condition of the subject,
and the weight
and metabolism of the subject. For example, when administered to a subject
suffering
from IC or AD, a tablet, pill, dragee, capsule, gelcap, troche, or capsule,
will contain an
amount of L-arginine andlor an HMG-CoA reductase inhibitor effective to,
ll2tel' alia,
3o ameliorate the harmful effects of insufficient blood flow to normal tissue,
i.e., prevent
the development of or alleviate the existing symptoms of, or prolong the
survival of, the
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subject being treated. Determination of an effective amount is well within the
capabilities of those skilled in the art, especially in light of the detailed
disclosure herein.
Therapeutically effective amounts for use in humans can also be estimated from
animal models. For example, a dose for humans can be formulated to achieve a
concentration found to be effective in animals.
A therapeutically effective dose can also be estimated from human
pharmacokinetic data. While not intending to be bound by any particular
theory, it is
believed that efficacy is related to a subject's total exposure to an applied
dose of
administered drug, and/or an active metabolite thereof, as determined by
measuring the
to area under the blood concentration-time curve (AUC). Thus, a dose
administered
according to the methods of the invention that has an AUC of administered
compound
(and/or an active metabolite thereof) within about 50% of the AUC of a dose
known to
be effective for the indication being treated is expected to be effective. A
dose that has
an AUC of administered compound (and/or an active metabolite thereof) within
about
70%, about 80% or even about 90% or more of the AUC of a known effective dose
is
preferred. Toxicity and therapeutic efficacy of such agents can be determined
by
standard pharmaceutical procedures in cell cultures or experimental animals,
e.g., for
determining the LD50 (the dose lethal to 50% of the population) and the ED50
(the dose
therapeutically effective in 50% of the population). The dose ratio between
toxic and
2o therapeutic effects is the therapeutic index and can be expressed as the
ratio LD50/ED50.
Formulations that exhibit large therapeutic indices are preferred. While
formulations
that exhibit toxic side effects may be used, care should be taken to design a
delivery
system that targets such formulations to the site of affected tissue in order
to minimize
potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used
in
formulating a range of dosage for use in humans. In one embodiment, the dosage
of
such formulations of the instant invention lies within a range of circulating
concentrations that include the ED50 with little or no toxicity. The dosage
may vary
within this range depending upon the dosage form employed and the route of
3o administration utilized. For any formulation used in the therapeutic or
prophylactic
methods of the invention, the therapeutically effective dose can be estimated
initially
from cell culture assays. A dose may be formulated in animal models to achieve
a
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circulating plasma concentration range that includes the IC50 (i.e., the
concentration of
the test compound which achieves a half maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more accurately
determine
useful doses in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
Adjusting the dose to achieve maximal efficacy in subjects based on the
methods
described above, particularly on the blood concentration and duration of
administered
compound and/or its active metabolites is well within the capabilities of the
ordinarily
skilled artisan.
III. Methods of Manufacture
It has been discovered that efficient and substantial incorporation or
coverage of
L-arginine granules within a matrix improves the sustained release
characteristics of the
compositions of the present invention. In the case of a cellulosic matrix,
upon contact
with water, the matrix is partially hydrated, forming a gel layer that
controls the rate of
release of the L-arginine. Efficient coating or incorporation of the L-
arginine granules
creates a temporary barner to dissolution that prolongs the delivery of the L-
arginine.
Substantial gaps in the matrix allow the L-arginine to dissolve too quickly.
The methods
of the present invention result in a product with improved properties versus
products
2o made by direct compaction. Further, the present method is advantageous over
methods
that include fluidization dispersions as these methods are time-consuming and
expensive.
The key to effective and efficient coverage is in performing the granulating,
milling, and blending steps of the present invention. Refernng to Figure 5, in
a preferred
embodiment, tablets are manufactured according a method that includes the
steps of
granulating the L-arginine (step 110), milling the L-arginine (steps 125,
140), blending
the L-arginine with the remainder of the ingredients (steps 145, 150, 155),
and
compressing the ingredients to form a tablet (step 160). Preferably, the
method also
includes either or both of the steps of screening the ingredients (step 105),
andlor drying
3o the L-arginine during the milling step (step 135).
If the ingredients are screened prior to use (step 105), a #20 and/or a #30
mesh
screen can be used for some or all of the ingredients. In a preferred
embodiment, the
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granules are screened before granulation (step 105), and again before milling
(not
shown). Screening provides granules with a narrower particle size distribution
in a range
that is advantageous for coating and/or compaction.
The step of granulating is advantageous in that it provides more uniform
particles. An active agent can be pelletized or granulated using any suitable
methods
known in the art. Pelletization or granulation is commonly defined as a size-
enlargement
process in which small particles are gathered into larger, permanent
aggregates in which
the original particles can still be identified and renders them into a free
flowing state.
Prior to granulation, a binder can be added to the active agent to improve the
granulation
1o process. Other additives can be added during granulation. These include,
e.g.,
sweeteners, flavors, color agents, antioxidants, etc.
Optionally, water or other solvent can be added to aid the granulation
process.
The amount of water or solvent added depends on, for example, the selection of
a
granulation process, and is readily determinable by those of skill in the art.
Water or
other solvent may be added at any suitable time point during the granulation
process.
For example, a binder may be mixed with a solvent (e.g., water) to form a
granulating
agent, and then the granulating agent can be sprayed onto active agents.
Alternatively, if
a granulating agent is too viscous to be uniformly sprayed onto active agents,
it may be
desirable to blend the binder with the active agent first and then spray water
or other
solvent to produce a uniform pattern of active agent granules or pellets.
Any suitable granulation method can be used to produce particles comprising an
active agent. Wet granulation and/or dry granulation methods can be used.
Dry granulation refers to the granulation of a formulation without the use of
heat
and solvent. Dry granulation technology generally includes slugging or roll
compaction.
Slugging consists of dry-blending a formulation and compressing the
formulation into a
large tablet or slugs on a compressing machine. The resulting tablets or slugs
are milled
to yield the granules. Roller compaction is similar to slugging, but in roller
compaction,
a roller compactor is used instead of the tableting machines. See, e.g.,
Handbook of
Pharmaceutical Granulation Technology, D. M. Parikh, eds., Marcel-Dekker, Inc.
pages
102-103 (1997). The dry granulation technique is useful in certain instances,
for
example, when the active agent is sensitive to heat or solvent.
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Alternatively, wet granulation can be used. In wet granulation, solvents and
binders are typically added to a formulation to provide larger aggregates of
granules.
The temperature during granulation can be set at any suitable point, generally
not
exceeding the melting point of any components of the formulation. Typically,
the
mixture is granulated at a temperature of about 35° C to about
65° C for about 20 to
about 90 minutes. In a preferred embodiment, the mixture is granulated for
less than
about 20 minutes, more preferably for about 1 to about 10 minutes at room
temperature
(see, Example ~). Then the granules are typically air dried for a suitable
duration (e.g.,
one or more hours).
l0 Preferably, the active agents are granulated by high shear mixer
granulation
("HSG") or fluid-bed granulation ("FBG"). Both of these granulation processes
provide
enlarged granules or pellets but differ in the apparatuses used and the
mechanism of the
process operation. These granulation techniques can be performed using
commercially
available apparatuses.
In HSG, blending and wet massing are accomplished by high mechanical
agitation by an impeller and a chopper. Mixing, densification, and
agglomeration of
wetted materials are achieved through shearing and compaction forces exerted
by the
impeller. The primary function of the chopper is to cut lumps into smaller
fragments and
aid the distribution of the liquid binder. The liquid binder is either poured
into the bowl
or sprayed onto the powder to achieve a mare homogeneous liquid distribution.
On the other hand, fluidization is the operation by which fine solids are
transformed into a fluid-like state through contact with a gas. At certain gas
velocities,
the fluid will support the particles, giving them freedom of mobility without
entrainment. Such a fluidized bed resembles a vigorously boiling fluid, with
solid
particles undergoing extremely turbulent motion, which increases with gas
velocity.
Fluidized bed granulation is thus a process by which granules are produced in
a fluidized
bed by spraying a binder solution onto a fluidized powder bed to form larger
granules.
The binder solution can be sprayed from, for example, a spray gun positioned
in any
3o suitable manner (e.g., top or bottom). The spray position and the rate of
spray may
depend on the nature of the active agent and the binder used, and are readily
determined
by those skilled in the art.
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In a preferred method according to the invention, granulating the L-arginine
(step
110) includes the steps of premixing the L-arginine with a binder such as
povidone to
form a blend (step 115), and granulating the blend with a granulating agent
(granulating
vehicle) in a granulator (step 120). The granulating agent can be, e.g.,
povidone
dissolved in purified water. Preferably, a high-shear granulator such as a
Niro PMA 65
High Shear Granulator is employed. The granulator can be used both to mix the
L-
arginine and binder, and also to granulate the blend while spraying the
granulating
vehicle on the blend.
After the granulation of one or more components of the formulation,
optionally,
to the granulated formulation can be milled. Milling can be performed using
any suitable
commercially available apparatus (e.g., CoMil equipped with a 0.039 inch
screen). The
mesh size for the screen can be selected depending on the size of the granules
desired.
After the granulated active agents are milled, they may be further dried
(e.g., in the air) if
desired.
i5 In a preferred embodiment, milling the L-arginine includes the steps of
milling
the wet granules or wet milling (step 125), drying the granules (step 130),
and milling
the dry granules or dry milling (step 140), in accordance with techniques well
known in
the art (see generally, U.S. Pat. No. 5,145,684 and European Patent
Application 498,482,
the contents of both of which are hereby incorporated by reference). A mill
such as a
2o CoMil can be employed to wet mill and dry mill the granules. In one
embodiment, the
mill is equipped with a '375Q screen for wet milling and a'062R screen for dry
milling.
The drying step can be accomplished by drying the granules in a bed dryer,
e.g., an
Aeromatic S-2 Fluid Bed Dryer, to a~ desired Loss on Drying (LOD) level, e.g.,
a < 3%
LOD. The drying steps can be accomplished in stages (step 135) until the
desired LOD
25 is reached.
Blending the L-arginine with the remainder of the ingredients can include a
pre-
blending step (step 145), a blending step (step 150), and a final blending
step (step 155).
The pre-blending step can include blending the L-arginine/povidone granules
with a
filler and a glidant, e.g., microcrystalline cellulose and colloidal silicon
dioxide. The
30 pre-blending step can be accomplished, e.g., in an 8 quart V-Blender, by
blending for
about 5 minutes at 25 rpm. The blending step can include adding to this blend
one or
more sustained release agents, e.g., one or more hydroxypropyl
methylcelluloses, and a
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filler, e.g., microcrystalline cellulose. The blending step can be
accomplished, e.g., in a
2 cubic foot V-Blender, by blending for about 20 minutes at 25 rpm. The final
blending
step can include adding a release agent/lubricant, e.g., magnesium stearate,
to the blend
in the 2 cubic foot V-blender and blending for about 5 minutes at 25 rpm.
After preparing the formulation as described above, the formulation is
compressed (step 160) into a tablet form. This tablet shaping can be done by
any
suitable means, with or without compressive force. For example, compression of
the
formulation after the granulation step can be accomplished using any tablet
press (e.g., a
Manesty Beta Press equipped with a 0.74" x 0.3 ~0" oval shaped, convex, plain
l0 tooling), preferably if the formulation composition is adequately
lubricated with
lubricant (e.g., magnesium stearate). Many alternative means to effect this
step are
available, and the invention is not limited by the use of any particular
apparatus. The
compression step can be carried out using a rotary type tablet press. The
rotary type
tableting machine has a rotary board with multiple through-holes, or dies, for
forming
15 tablets. The formulation is inserted into the die and is subsequently press-
molded.
Alternatively, the tablets can be made by molding. Molded tablets may be made
by molding in a suitable machine a mixture of the powdered compound moistened
with
an inert liquid diluent.
The diameter and shape of the tablet depends on the molds, dies and punches
2o selected for the shaping or compression of the granulation composition.
Tablets can be
discoid, oval, oblong, round, cylindrical, triangular, and the like. The
tablets may be
scored to facilitate breaking. The top or lower surface can be embossed or
debossed with
a symbol or letters.
The compression force can be selected based on the type/model of press, what
25 physical properties are desired for the tablet product (e.g., desired
hardness, friability,
etc.), the desired tablet appearance and size, and the like. Typically, the
compression
force applied is such that the compressed tablets have a hardness of at least
about 2 kp.
These tablets generally provide sufficient hardness and strength to be
packaged, shipped
or handled by the user. If desired, a higher compression force can be applied
to the
30 tablet to increase the tablet hardness. However, the compression force is
preferably
selected so that it does not deform (e.g., crack or break) the active agent-
containing
particles within the tablet. Preferably, the compression force applied is such
that the
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compressed tablet has a hardness of less than about 10 kp. In certain
embodiments, it
may be preferred to compress a tablet to a hardness of between about 3 kp to
about 7 kp,
optionally between about 3 kp to about 5 kp, or about 3 kp.
Typically, the final tablet will have a weight of about 50 mg to about 2000
mg,
more typically about 200 mg to about 1000 mg, or about 400 mg to about 700 mg.
The particular formulation and methods of manufacturing the formulation of the
present invention impart unique advantages on the sustained release L-arginine
composition. In particular, the formulation and the methods of the present
invention
render a composition that achieves a desirable sustained release dissolution
profile.
1o Optimally, a sustained release L-arginine formulation would sustain ih
vitro drug release
at least up to 14 hours, preferably about 10% to about 40% at about 1 hour,
about 30% to
about 70% at about 4 hours, about 55% to about 75% at about 6 hours, about 65%
to
about 85% at about 8 hours, about 75% to about 95% at about 12 hours and about
80%
to about 100% at 14 hours. As demonstrated by Figure 7, the formulation of the
present
invention achieves such optimal dissolution. Furthermore, as shown in Example
8 and
Example 14, dissolution and stability studies demonstrate that the formulation
of the
present invention displaxs an optimal dissolution profile one and two months
following
manufacturing.
Furthermore, the formulation and methods of the present invention render a
2o sustained release L-arginine composition that is not excessively friable.
Furthermore the
formulation and methods of the present invention render a sustained release L-
arginine
composition that is sufficiently compressible to allow for convenient
manufacturing of
the composition.
If desired, other modifications can be incorporated into embodiments of the
tablet. For example, modification of active agent release through the tablet
matrix of the
present invention can also be achieved by any known technique, such as, e.g.,
application
of various coatings, e.g., ion exchange complexes with, e.g., Amberlite IRP-
69. The
tablets of the invention can also include or be coadministered with GI
motility-reducing
drugs. The active agent can also be modified to generate a prodrug by chemical
3o modification of a biologically active compound that will liberate the
active compound in
vivo by enzymatic or hydrolytic cleavage, etc. Additional layers or coating
can act as
diffusional barners to provide additional means to control rate and timing of
drug
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release.
If an HMG CoA-reductase inhibitor (e.g., simvastatin) and/or additional agents
are included, preferably these agents are added in the blending steps (steps
145, 150,
155). When the tablet comprises a sustained release L-arginine formulation and
an
HMG-CoA reductase inlubitor~formulation, the tablet may have a core of slow
release L-
arginine formulation and a second outer cover or coating of a formulation
comprising at
least one HMG-CoA reductase inhibitor. Alternatively, the tablet may comprise
an L-
arginine formulation, e.g., a sustained release L-arginine formulation, and a
HMG-CoA
reductase inhibitor formulation sharing one surface.
When L-arginine is administered either sequentially or concurrently with HMG-
CoA reductase inhibitors, each tablet, cachet, troche, or capsule contains
from about 0.01
mg to about 200 mg of the HMG-CoA reductase inhibitors. The amount of an HMG-
CoA reductase inhibitor will vary depending on the particular HMG-CoA
reductase
inhibitor utilized.
In another aspect of the present invention, a composition for the treatment of
cardiovascular and/or cerebrovascular disease is provided in the form of food.
Preferably, the food is in the form of a bar such as a prescription health
bar. Use of food
enables the provision of larger amounts of L-arginine than could be
incorporated into a
single tablet, e.g., it is difficult to incorporate more than 1 gram of L-
arginine in a single
tablet. Thus, multiple tablets are required for delivery of amounts of L-
arginine in
excess of 1 gram. The present invention provides a bar that can provide more
than 1
gram of L-arginine as well as other agents, as desired. In one embodiment, the
L-
arginine is added as an immediate release formulation, e.g., immediate release
granulars
of L-arginine, to a food bar. Preferably, 'the bar includes a sustained
release formulation
that includes, for example, sustained release granulars of L-arginine. hz a
preferred
embodiment, the granulars include taste masking constituents, e.g., taste
making
coatings. In another embodiment, the bar further contains additional agents,
such as an
HMG-CoA reductase inhibitor. Preferably, the HMG-CoA reductase inhibitor, is a
statin
such as simvastatin. Combining L-arginine with statins in a food vehicle form
would
provide continence and an easy to administer the formulation. Use of food also
can
reduce the deed for taking multiple tablets of L-arginine when a higher dose
is desired.
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In one embodiment, the bars have between about 1 and about 80 g of simvastatin
and 'between about 1 and about 10 grams of L-arginine. In a preferred
embodiment, bars
are provided having a total of at least about 10 mg of simvastatin and about 4
g per bar
of L-arginine or its salts in conjunction with sugars, fruit components,
protein, and
vitamins and minerals. The bar weighs in the range of about 25 to about 100 g.
In a
particular process, the bar is produced by combining sugars and fruit paste at
an elevated
temperature and then combining the syrup at a reduced temperature with the
minor
ingredients. After blending the minor ingredients in the syrup, the L-arginine
and the
simvastatin are added, particularly in conjunction with a protein extender,
followed by
1o bulking and food agents, particularly fruit pieces or other particulate
edible ingredients
providing the desired texture and flavor, and soy proteins. The resulting
product is
storage stable, has desirable organoleptic properties in being tasty, and
provides a
healthy combination of ingredients in collaboration with the simvastatin and L-
arginine.
Methods and formulations for manufacturing health bars with L-arginine and L-
lysine
are described in, e.g., U.S. Patent No. 6,063,432, incorporated in its
entirety by this
reference.
Another aspect of the present invention is a method of manufacturing the bar
described above. The method would include granulating the L-arginine as
described
above in connection with Figure 5, step 110. Preferably the granulating step
would
2o include the pre-mixing step (step 115) and the granulating step (step 120).
Preferably,
the method also includes the wet milling step (step 125) described above. Such
bar
would be obtained by wet granulation of the L-arginine with appropriate
excipients, such
as detailed above. The resulting granulars would be either used as is or be
coated with
taste masking cellulosics.
This invention is further illustrated by the following examples that should
not be
construed as limiting. The contents of all references, patents and published
patent
applications cited throughout this application are incorporated herein by
reference.
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EXAMPLES
EXAMPLE 1: Tablet Formulation 1
About 250 grams of L-arginine was placed in a mixer and as it was slowly mixed
at 100 RPM, 100 g EUDRAGIT RS 30D low permeability methacrylic aqueous polymer
dispersion (Rohm America, Piscataway, NJ) was added to form a wet mass. The
wet
mass was passed through 18-20 sieves and allowed to dry at 50°C for 24
hours. The
resulting dry L-arginine granulars (250 g) were dry mixed with 84 g METHOCEL
K100
M CR methylcellulose (The Dow Chemical Company, Danbury, CT) and 3 g magnesium
l0 stearate to form a blend. The resulting blend was compressed into tablets
using 7/16
concave punches.
EXAMPLE 2: Tablet Formulation 2
250 g of L-arginine was placed in a mixer and as it was slowly mixed, 84 g
METHOCEL K100 M CR methylcellulose and 3 g magnesium stearate were added. The
resulting blend was compressed into tablets using 7/16 concave punches.
EXAMPLE 3: Capsule Formulation 1
250 g L-arginine was placed in a mixer and as it was slowly mixed, 100 g
2o EUDRAGIT RS 30D low permeability methacrylic aqueous polymer dispersion was
added to form a wet mass. The wet mass was passed through 18-20 sieves and
allowed
to dry at 50°C for 24 hours. The resulting dry L-arginine granulars
(250 g) were dry
mixed with 84 g METHOCEL K100 M CR methylcellulose and 3 g magnesium stearate
to form a blend. The resulting blend was placed into 00 gel capsules.
EXAMPLE 4: Capsule Formulation 2
250 g L-arginine was placed in a mixer and as it was slowly mixed, 84 g
METHOCEL K100 M CR methylcellulose and 3 g magnesium stearate were added. The
resulting blend was placed into 00 gel capsules.
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EXAMPLE 5: Tablet Formulation 3
250 g L-arginine and 50 g METHOCEL K100 M CR methylcellulose were
mixed and homogenized using a Kitchen Aid~ mixer on low speed for 10 minutes
to
form a dry blend. To the dry blend, 115 g EUDRAGIT RS 30I~ low permeability
methacrylic aqueous polymer dispersion was added in 5 g increments until the
mass was
homogeneously wet. The wet mass was passed through a 12 mesh sieve followed by
a
20 mesh sieve and subsequently, allowed to dry at 30°C for 24 hours
until the moisture
content was 1 % by weight. The resulting dry L-arginine granulars were dry-
mixed with
7 g magnesium stearate and then compressed, using a Beta Manesy press, into
tablets
using 7/16 concave punches.
EXAMPLE 6: Manufacturing of a Sustained Release Tablet
About 1000 g L-arginine and, about 200 g METHOCEL K100 M CR
methylcellulose were mixed in a GP-1 high shear mixer (granulator) for about 5
minutes
at 100 RPM. About 138 g EUDRAGIT RS 30I~ low permeability methacrylic aqueous
polymer dispersion was then added with the impeller running at 200 RPM and a
pressure
of 1.5 bar. The mixture was granulated for 1 minute at 200 RPM. The
granulation was
then dried in an MP-1 Fluid Bed Granulator at 45°C inlet temperature
with an air flow of
100 CMH to approximately 2% moisture content. The dried granules were then
milled
using a Comil 1975 with size SSR screen and round impeller at 90% speed. In an
8 Qt.
V-Blender, about 27 g magnesium stearate was added to the milled granules and
mixed
for 2 minutes. The material was then compressed into tablets with a target
weight of
682.5 mg to highest possible hardness using a Beta Manesty Press with 7/16"
standard
concave tooling. The tablets were hand-packaged at 60 tablets per bottle in 75
cc HDPE
Bottles.
The release profile of the tablet versus commercially available sustained
release
L-arginine tablets purchased from BioEnergy (Warren, NJ), was generated using
high
performance liquid chromatography (HPLC). Figure 7 is a chart depicting the
release
profiles of both formulations. '
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EXAMPLE 7: Evaluation of Pharmacokinetics of L-arginine
A randomized, four-way crossover study to evaluate the pharmacokinetics of L-
arginine sustained release tablets versus immediate release capsules was
conducted on
14 healthy adult volunteers under fasting conditions. "Healthy" as used herein
means
nonhypercholestermic subjects with no cardiovascular risk factors. The study
compared
the sustained release L-arginine tablet (L-arginine SR) of Example 6 and
commercially
available immediate release L-arginine capsules (L-arginine IR) purchased from
Montiff
(Los Angeles, CA).
The study goal was to determine the pharmacokinetic parameters of sustained
to release L-arginine. As depicted in Table I below, based on the p-values
from the two-
tailed paired t-test performed on each pharmacokinetic parameters, there was a
statistically significant difference between treatments for Cm~ and Tm~. As
expected,
sustained release L-arginine tablets had a lower CmaX (14.9 ug/mL versus 24.1
ug/mL)
and a longer T",~ (4.4 h versus 1.4 h) compared with the immediate release
capsules.
Table I: PIE Parameters of L-arginine SR v. L-arginine IR
L-ar mine CmaX AUCo_t AUCo_lo TmaX Tm~x o-10
o-t
L-Arg SR 14.9 143 68.56 4.4 3.27
L-Arg IR 24.1 147 92.23 1.4 1.35
Ratio 0.62 0.97 0.74 3.2 2.43
P-value 0.0005 0.677 0.0382 0.0133 0.0073
EXAMPLE 8. Manufacturing of an Improved Sustained Release L-arginine Tablet
Table II lists the ingredients assembled to manufacture an improved sustained
2o release tablet, as well as the amounts used of each ingredient.
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Table II: Ingredients
Component mg/ tabletPercoentageWeight/ Batch
/o K
L-ar 'nine monohydrochloride 500 50 12.5
Povidone (K 29/32) 35 3.5 0.88
Purified Water - - 2*
Hydroxypropyl Methylcellulose275 27.5 6.87
(METHOCEL K100M P CR
Hydroxypropyl Methylcellulose
75 7.5 1.88
(METHOCEL E 4M CR)
Microcrystaline Cellulose 102 10.2 2
5 56
AVICEL PH 102 . .
Colloidal Silicon Dioxide 5 0.5 0.13
Ma esium stearate 7.5 0.75 0.18
TOTAL: ~ 1000 ~ 100.0 25
~
*Water is used in granulation and then the mixture was dried
All ingredients, except the magnesium stearate, were screened in a #20 mesh
screen. The magnesium stearate was screened in a #30 mesh screen.
Approximately
half of the povidone (polyvinylpyrrolidone) was dissolved in purified water
and set aside
as a granulating agent. The L-arginine and the remainder of the povidone were
dry
mixed for 4 minutes in a Niro PMA 65 High Shear Granulator, and then
granulated for
about 6.5 minutes by spraying the granulating agent into it. The wet granules
were then
l0 milled in a CoMil mill equipped with a'375Q screen. The milled granules
were then
dried in an Aeromatic S-2 Fluid Bed Dryer to a LOD of _< 3%. The dxied
granules were
then milled in the CoMil equipped with a'062R screen. Approximately half of
the
microcrystalline cellulose and the collodial silicon dioxide were then blended
in an 8
quart V-Blender for 5 minutes at 25 rpm and transferred to a 2 cubic foot V-
Blender.
The remaining portion of the microcrystalline cellulose and the hydroxylpropyl
methylcellulose were then also added to the 2 cubic foot V-Blender and blended
for 20
minutes at 25 rpm. The magnesium stearate was then added to the 2 cubic foot V-
Blender and blended for 5 minutes at 25 rpm. Finally, the blend was compressed
into
tablets with a target weight of 1000 mg using a Manesty Bet Press equipped
with 0.748"
x0.380" oval shaped, convex, plain tooling. Figure 6 is a schematic flow
diagram of
this method.
;Standard in-process controls tests and specifications can be used during the
manufacturing process, the ones used for this example are listed in Table III
below.
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Table III: L=ar~inine SR Tablets Inprocess Controls: Specifications and
Methods
S ecification: Method Acce tance Criteria
Mean: 90.0% - 110.0% of
Label
Blend UniformityCTMLP-663 Claim
RSD% NMT 5.0%
Bulk & Ta DensitySOP Lab 2010 Re ort results
Particle Size SOP LAB 201 Report results
~
Distribution
Moisture SOP Lab 2059 NMT 3.5%
Standard release methods and specifications can be used, the ones used for
this
example are provided in Table IV below.
Table 1V: L-ar~inine SR Tablets Release Methods and Specifications
S ecification: Method Acce tance Criteria
Physical Visual White to off white tablets
A earance Ins ection Oval sha ed, convex tablet
The retention time and on-line
UV spectrum
Identification CTMPLP-663 (200-400 mn) of the sample, correspond
to
those of the reference standard
Potency CTMLP-663 90.0 -110.0% of label claims
~dividual: NMT 0.5%
Related SubstancesCTMLP-663 Total: NMT 2
0%
.
Moisture SOP LAB 2059 NMT 3.5%
1 hr 10 -40%
4 hr 30-70%
Dissolution CTMLP-663 12 hr > 75/~
Profile
Record Profile
Content UniformiCTMLP-663 USP <905>
Total Aerobic Microbial count <100
cfu/mL
Total Combined Molds and Yeast
count <50
cfu/mL
Microbial LimitsUSP <61> Absence of E. coli
Absence of S. aureus
Absence of P. aeruginosa
Absence of Salmonella s ecies
l0
Furthermore, the studies have demonstrated desirable physical characteristics,
including friability and content uniformity for the sustained release L-
arginine
formulations of the present invention.
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Table V: Physical Testing, Potency, Content Uniformity and Dissolution for
Two batches of the SR L-ar~inine formulation
Batch # 1 2
Tablet t ' 1 a n =2 .70
0
0
ontent 'f rmi n = ~ o .0 0.
0
0
0
7 .6
0.3 0.
14 98.4 92.5
E~~AMPLE 9: Evaluation of pharmacokinetics of L-arginine SR with and
5 without Simvastatin and Simvastatin with and without L-arginine
SR
The pharmokinetics of L-arginine SR with and without simvastatin, and
simvastatin with and without L-axginine SR were studied. The L-arginine SR
tablets of
to Example 6 were used as well as commercially available simvastatin tablets
purchased
from BioEnergy (Warren, NJ).
As can be see in Table VI, based on the p-values from the two-tailed paired t-
test
performed on each pharmokinetic parameter, there was not a statistically
significant
difference between treatments for Cmax, AUCo-io, and TmaX. As depicted in
Table VII, L-
arginine SR has no statistically significant effect on the single dose
pharmokinetics of
' simvastatin.
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Table VI. L-ar,:;inine PK Paramaters with and without Simvastatin
L-arginine Cmax AUCo_lo Tmax
(mg/ml) (mg- (hr)
hr/ml)
L-Arg SR 14.77 68.56 3.27
L-Arg SR with 13.49 51.55 3.23
Simvastatin
Ratio ~ 1.09 1.33 1.01
P-value 0.5001 0.0713 0.9716
Table VII. Simvastatin PK Paramaters with and without L-ar i~ nine
Simvastatin Cmax AUCo_to Tmax kelim t 1/2
(n /ml) (n -hr/ml)(hr) ( 1 /hr) (hr)
simvastatin 21.15 107.93 2.68 0.1248 6.56
w/o
L-arginine
SR
simvastatin 18.95 114.36 2.29 0.0950 10.01
with
L-arginine
SR
P-value 0.5360 0.6302 0.4758 0.1526 0.1059
1o EXAMPLE 10: Effect of Administration of Simvastatin with L-arginine Upon
Infarct Size in Mice
The effect of administration of both simvastatin and L-arginine upon infarct
size
was studied in mice. Mice were given interperitoneal injections comprising
simvastatin,
and simvastatin and L-arginine, dissolved in saline solution in the amounts
indicated in
Figure 3. The results of infarct size on these mice versus a control group are
depicted in
Figure 2 and Figure 3.
EXAMPLE ,11: Dose Optimization of Combination of Simvastatin and L-arginine
Dose optimization of combined administration of simvastatin and L-arginine was
studied in mice. Mice were injected with varying levels of simvastatin and L-
arginine as
shown;in Figure 4. The results of this study are also shown in Figure 4.
Statistical
analysis predicted that the optimal range of the combination to be 1.2-1.4
mg/Kg .
simvastatin with about 20-25 mg/Kg L-arginine.
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EXAMPLE 12: Improvement of Endothelium-dependent Vasodilation by
Simvastatin is Potentiated by Combination with L-arginine
Sustained Release in patients with Elevated ADMA Levels
Statins stimulate the expression of endothelial NO synthase (eNOS) in vitro
and
enhance endothelium-dependent, NO-mediated vasodilation in vivo. Asymmetrical
dimethylarginine (ADMA) is an endogenous, competitive inhibitor of eNOS. The
presence of elevated plasma ADMA levels is associated with endothelial
dysfunction. It
to was discovered that simvastatin enhances endothelial function in patients
with elevated
ADMA only if the inhibitory effect of ADMA is overcome by supplemental L-
arginine
sustained release.
clinically asymptomatic, elderly subjects with elevated ADMA levels
received, in a randomized order, simvastatin (40~ mg/day), L-arginine
sustained-release
15 (3 g/day) prepared as described in Example 8, or a combination of both,
each for 3
weelcs, in a three period crossover design with at least three weeks of wash-
out between
treatments. Endothelium-dependent vasodilation was assessed by brachial artery
ultrasound using computer-assisted image analysis; ADMA and L-arginine plasma
concentrations were determined by a validated HPLC method.
2o Analysis of 15 patients who completed the study revealed that both
sustained
release L-arginne alone or in combination with simvastatin increased
percentage
endothelial-dependent vasodilation, from pre-treatment measurements. The
combination
significantly increased the change from pre-treatment percentage endothelial-
dependent
vasodilation by 3.87% over that observed with simvastatin alone (p<0.025). The
difference in the change in percentage endothelial-dependent vasodilation
between the
combination and sustained release L-arginine alone was small. Endothelium-
independent vasodilation by glyceryl trinitrate was not affected by any of the
treatments.
L-arginine sustained release, either alone or in combination with simvastatin,
significantly improved plasma L-argininelADMA ratio (baseline, 82.34.0 vs.
102.89.2
3o and 102.610:8, respectively, each p<0.05). These results are summarized in
Figure 8.
Simvastatin does not enhance endothelial function in subjects in whom eNOS is
blocked by elevated ADMA levels; combination of simvastatin with oral L-
arginine
sustained release has a synergistic effect on endothelial function. As NO-
mediated
effects may play a major role in therapeutic effects of statins, combination
with L-
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arginine sustained release should be considered in patients with elevated ADMA
concentration.
EXAMPLE 13: Improvement in Cholesterol Levels by Treatment with
Simvastatin in Combination with L-arginine Sustained
Release
In the study described in Example 12, the change in total cholesterol (TC),
LDL
cholesterol, HDL cholesterol, and triglycerides was analyzed pre- and post-
treatment.
to The results of this analysis are shown in Figure 9. As the results
demonstrate, the co-
administration of sustained release L-arginine of the present invention and
simvastatin
lowers the total cholesterol, LDL cholesterol and triglycerides, and increases
the HDL
cholesterol to a greater degree than administration of simvastatin alone.
EXAMPLE 14: Determination of Dissolution Release of Arginine HCl in
Sustained Release Arginine HCl 500 mg Tablets by HPLC
The mobile phase was prepared as follows. Initially, one liter of pH 3.3
buffer
solution was prepared by weighing about 0.9 g of 1-pentanesulfonic acid sodium
salt,
monohydrate and 3.5 g of sodium phosphate monobasic, monohydrate into a
suitable
container. About 100 mL of deionized water was added to dissolve. The pH was
adjusted to 3.3 by the addition of phosphoric acid. Subsequently, 850 mL of
the pH 3.3
buffer was combined with 150 mL of methanol into a suitable container and
mixed. The
mixture was filtered through a 0.45 wm nylon membrane filter. Finally the
mixture was
degassed before use.
The dissolution medium (50 mM phosphate buffer at a pH of 6.8) was prepared
as follows. Ziutially 20.0 mL of 10 M NaOH was pipetted into a 1000 mL
volumetric
flask and diluted with deionized water to prepare 0.2 M NaOH. Subsequently
54.44 g of
Potassium Dihydrogen Phosphate, Anyhydrous was weighed into a suitable
container,
3o and dissolved and diluted with 2000 mL of deionized water. 896 mL of the
0.2 M
NaOH was added to the container and diluted to 8000 mL with deionized water.
Finally
the mixture was degassed before use.
The dissolution sample was prepared as follows. Six Arginine HCl 500 mg
tablets, prepared as described in Example 8, were weighed. Each tablet was
placed in a
stainless steel sinlcer with 900 mL of Phophate 'buffer (pH 6.8). The sinlcer
was
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subsequently dropped into a vessel of a USP Apparatus 2 (paddle) for immediate
rotation at 75 rpm at about 37° C ~ 0.5° C. 10 mL of the
solution from the vessel was
removed at 1, 2, 4, 6, 8, 10, 12 and 14 hour time points for respective
dissolution
analysis at each time point. Each of these samples solutions were filtered
through 0.45
~m PVDF syringe filters. The filtrate was collected into HPLC vials for
analysis,
wherein the first 1-2 mL were discarded. Using a 10 ~,m Full Flow Filter, 10
mL of the
dissolution mediu'rn pre-warmed to 37° C ~ 0.5° C was replaced
back to the dissolution
vessel after every sampling point. The practitioner should be aware that the
sample
solution is stable up to 1 day at room temperature and is stable up to 3 days
at 4° C.
l0 The Arginine HCl standaxd solution was prepared as follows. 28 ~ 2 mg of
Arginine HCl reference standard is accurately weighed into a 50 mL volumetric
flask.
The standard was dissolved in and diluted to volume with dissolution medium.
HPLC was conducted using a BDS Hypersil C18 column (5 Vim, 250 mm x 4.6
mm) detecting using UV at 210 nm. The column temperature was set to ambient.
Generally, the run time was 9 minutes, the injection volume was 10 wL, the
flow rate
was 0.8 mL/min and the mobile phase was pH 3.3. Buffer/Methanol (85/15, v/v),
prepared as described above.
Each trial proceeded as follows. One injection of dissolution medium followed
by five consecutive injections of Arginine HCl standard solution and finally
one
2o injection of each sample solution were performed. Arginine HCl standard
solution was
reinjected after every six sample injections and at the end of the sequence
run. The
system drift throughout the run (i.e., the percent recovery of the standard
solution
compared to the mean of five consecutive injections of Arginine HCl standard
solution)
should be from about 97% to about 103%.
In determining the percent of arginine released, the practitioner must be
careful to
ensure that the USP trailing factor (T) for Arginine HCl peak in the injection
of working
standard solution is less than 2. T is calculated as follows:
T= w.os/2f
where W,os is the peak width of Arginine HCl peak at 5% of the peak height
from the
baseline, and f is the distance from the peak maximum to the leading edge of
the peak
(the distance being measured at a point 5% of the peak height from the
baseline.
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The percent Arginine HCl released is calculated as follows:
n-1
Release = [(CS)(V)(R°/RS) + ~C;Vr]/(LC)
=i
where n is the total number of measurements, Vr is the volume of dissolution
medium for
each measurement (10 mL), V is the initial volume of dissolution medium (900
mL), CS
is the concentration, in mg/mL, of Arginine HCl in the Working Standard
Solution, C; is
the concentration, in mg/mL, of Arginine HCl in each sample solution (where,
i=1 to
i=n-1), R" is the peak area response of Arginine HCl peak obtained from the
sample
to solution, RS is the average peak area response of Arginine HCl peak
obtained from the
consecutive injections of Working Standard Solution, and LC is the label claim
of
Arginine HCl (500 mg).
The percent released was calculated at 1, 2, 4, 6, 8, 10, 12 and 14 hours.
Table
VI and VIII summarize the results for various dissolution studies.
Table VIII: Dissolution Profiles of L-ar~inine SR Tablets at about 40°
C/75%RH
Stability
0 0
10 .l
14 89.1 92.4 ~ 96.0
EXAMPLE 15: Simvastatin-dependent regulation of eNOS expression
2o The following protocol was used to investigate the mechanism of the
simvastatin
dependent increase in eNOS function using cultured human aortic endothelial
cells
(HAEC) to differentiate between de hovo protein synthesis versus protein
mobilization
or protein activation in the up regulation of eNOS function.
Human aortic endothelial cells (HAEC-c) (BioWhittaker, Walkersville, MD)
were cultured according to the following procedure. Endothelial cells in EBM-
2/EGM-2
media (BioWhittaker) were grown to about 80% to about 90% confluence. Each
flask of
cells was washed with 5 ml media followed by the addition of 15 ml .media to
each cell.
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Cells were detached with a cell scraper and transferred to a 50 ml conical
tube. Cells
were pelleted by centrifugation at 800 RMP for 8 min. The supernatant was
discarded
and the pellet was washed with cold lx PBS.
The cells were homogenized as follows. The pellet was loosened and 400 ~1 of
l Ox homogenization buffer (250 mM Tris at pH 7.4, 10 mM EDTA and 10 mM EGTA)
was added. The sample was homogenized using a 27G needle about 10 times. The
homogenate was transferred to a 1.5 ml epindorph tube. The pellet was
subsequently
resuspended in 30 to 45 ~1 of lx homogenization buffer.
The cells were assayed as follows. Resin slurry was prepared by washing Resin
AG 50W-X8 (BioRad Laboratories, Hercules, CA) in an appropriate size column
with 5
bed volumes of 0.5 N NaOH. The column was washed with 20 volumes of water. The
resin was equilibrated with stop/equilibration buffer (50 mM NaAcetate at pH
5.5) until
the eluate is within 0.05 pH units of the stop/equilibration buffer. The
resulting solution
is stored at 4° C as a 50% slurry in stop/equilibration buffer. In
addition, fresh l OmM
NADPH in 25 mM tris (pH 7.4) was prepared by adding 602 w1 tris to a 5 mg vial
of
preweighed NADPH. A 1 ~,M Calmodulin solution was prepared by adding 0.069 mg
calinodulin to 4.1 mL water. 8 ~M CaCl in water was also prepared. 2x reaction
buffer
was prepared by combining 50 mM Tris (pH 7.4), 6 ~M BH4, 2 ~,M flavin adenine
dinucleotide, and 2 ~M flavin adenine mononucleotide. Subsequently, reaction
mixture
2o for each sample was prepared by combining 25 ~,l 2x reaction buffer, 5 ~1
10 mM
NADP, 5 x,18 mM CaCl2, 4 ~,1 Calmodulin solution and 1 ~,114C Arginine. 40 ~,1
of
reaction mixture and 5 ~,1 of sample or controls were combined in a 1.5 ml
centrifuge
tube. The tube was incubated for 1 hour at 37° C.
Columns were prepared by initially cutting the tip from a 1 ml pipette tip to
increase the minimal diameter of the tip. 250 ~1 of resin slurry, prepared as
described
above, was pipetted into each column (Fisher Scientific, Glenlake, IL). The
columns
were washed twice with 400 ~1 stop/equilibration buffer (50 mM NaAcetate at pH
5.5)
Following incubation, about 400 ~,1 of stop equilibration buffer (50 mM
Na.Acetate at .pH 5.5) was added to each sample and control. 400 ~,l of this
mixture is
3o added to the equilibrated column. Each column was washed with 400 ~1
stop/equilibration buffer. 400 ~1 of column eluate was transferred to
scintillation vials
with 4 ml of scintillation fluid. The resulting solution is mixed well on a
vortexer. A
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scintillation counter (Beta counter, Beckman Coulter, Inc., Fullerton, CA) is
used to
obtain the desired counts. Results are calculated, in part, by subtracting
background
(buffer control) from each sample. The sample values are expressed as counts
per
minute or as a percent of untreated cells.
Relative eNOS function was measured by the conversion of labeled-L-arginine to
L-citrulline and expressed as a percent of citrulline produced by the non-
treated cells.
Figure 10 shows data from an experiment where HAEC were incubated with 1.0 or
.3
pM simvastatin for 24 hours prior to the determination of eNOS function.
Untreated
cells were cultured concurrently and used to calculate relative eNOS function.
Figure 10
l0 clearly demonstrates that simvastatin increases the level of eNOS function
in cultured
endothelial cells.
The collective data demonstrates that simvastatin effects eNOS expression and
function in endothelial cells. A requirement for protein synthesis in the up
regulation of
eNOS function and the simvastatin-dependent increase in both eNOS-specific
mRNA
15 and function are consistent with a model of drug-induced modulation of eNOS
gene
transcription.
Eguivalents
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, many equivalents to the specific embodiments of the
invention
2o described herein. Such equivalents are intended to be encompassed by the
following
claims.
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